ES2335368T3 - EML4-ALK FUSION GEN. - Google Patents

Abstract

The present inventors found that a fusion gene present in some cancer patients is an oncogene. The present invention relates to a polypeptide as a novel fusion protein, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a transformed cell comprising the vector, a method for detecting the fusion protein or polynucleotide, a method for screening a therapeutic agent for cancer, and a therapeutic agent for cancer. Further, the present invention relates kit, primer set, and probe useful in the detection of cancer that is shown to be positive for the fusion gene.

Description

EML4-ALK fusion gene.

Background of the invention Field of the Invention

The present invention relates to a polypeptide as a novel fusion protein, to a procedure to detect the fusion protein or a polynucleotide that encodes the polypeptide and related materials.

Prior art

So far several related genes are known with cancer In particular, it has been shown that the genes of tyrosine kinase, which encode important enzymes that regulate directly cell growth, they are activated even by substitution or deletion in amino acid sequences and thus cause carcinogenesis

For example, NPM-ALK, the genes Fusion encoding NPM fused with the ALK tyrosine kinase, it  observed in more than half of cases of anaplastic lymphoma of large cells (LACG) and it has been shown that the activation of the ALK kinase is important for tumor cell growth by NPM-ALK (non-patent documents 25 and 14).

The presence of a new type of abnormal kinase found in approximately 10% of cases of lung cancer and yet this report has not done reference to specific molecules (non-patent document 2).

In 2000 EML4 (protein 4 similar to protein associated with echinoderm microtubules) (document no patent 3). The EML4 protein has a basic region at the end amino, and additionally has WD domains at the carboxyl end (non-patent document 8). The functions are little known Physiological EML4.

On the other hand, ALK (lymphoma kinase anaplastic) (non-patent document 4) is tyrosine kinase recipient (non-patent document 5).

The full-length ALK expression has been reported so far in some cancer cells of origin ectodermal such as neuroblastoma, glioblastoma, breast cancer and melanoma (length ALK expression not observed complete in cancer cells of endodermal origins and mesodermal) (non-patent document 13). The length ALK Complete is expressed in many neuroblastoma cell lines. Without  However, ALK autophosphorylation is not observed in these lines. neuroblastoma cells. In addition, from the analysis of cohort of patients with neuroblastoma has been reported that the ALK expression is weakly associated with cancer. It has been suggested that ALK expression in neuroblastoma may reflect its expression in normal neural differentiation, instead of its association with cancer (non-patent document 10). On the other hand, in informed cases, ligands such as pleiotrophin and midcin, in addition to the gene amplification of ALK itself, increase the ALK autophosphorylation and mobilize intracellular signals. Too It has been reported that ALK can contribute to the growth of cancer cells (non-patent document 12).

In some cases of human malignant lymphoma and Myofibroblastic inflammatory tumor has been reported that the ALK gene is fused to other genes (NPM, CLTCL, TFG, CARS, SEC31L1, etc.) as a result of translocation or chromosomal inversion and so forms a type of tyrosine kinase fusion (non-patent documents 9, 11, 14 to 19 and 26 to 28). In addition, a procedure has been reported. to identify a protein as a fusion component for ALK using ALK antibodies (non-patent document 6). For other In part, an EML4 and ALK fusion gene has not been reported. Due that most molecules have a formation domain of complex, the fusion protein itself has not been thought to form a complex. It has been considered that this complex formation causes loss of control of the tyrosine kinase activity of ALK and induces carcinogenesis with abnormally intracellular signals activated (non-patent document 10). In fact, it has been reported that the use of ALK hRNA or ALK kinase inhibitor compound for lymphoma cells expressing ALK fusion proteins can induce cell growth inhibition and cell death. By Therefore, it has been suggested that the ALK fusion protein may serve as a therapeutic target for lymphoma and inflammatory myofibroblastic tumor  (non-patent documents 20 to 22). It has also been suggested that the ALK can serve as a therapeutic target for other cancers whose growth implies ALK as described above (documents of non-patent 21 to 22).

Marzec et al. have reported that WHI-P131 and WHI-P154 (both, EMD Biosciences), which have been used as inhibitors of JAK3 tyrosine kinase, inhibit the activity of NPM-ALK (non-patent document 22). Another group has reported that the low molecular weight ALK inhibitor induces the cell death of lymphoma cell lines that express NPM-ALK (non-patent document 21). Also up several low molecular weight compounds have now been reported that have an inhibitory activity against ALK (documents of no patent 23 and 24 and patent documents 1 to 3).

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[Patent document 1] Document pamphlet WO 2004/080980

[Patent document 2] Document pamphlet WO 2005/009389

[Patent document 3] Document pamphlet WO 2005/016894

[Non-patent document 1] "The New England journal of medicine ", (USA), 2005, vol. 353, p. 172-187

[Non-patent document 2] Proceedings of the 65th Annual Meeting of the Japanese Cancer Association, O-324 (granted on August 28, 2006)

[Non-patent document 3] registration number from GenBank: NM_019063

[Non-patent document 4] registration number from GenBank: AB209477

[Non-patent document 5] Oncogene. 30 of January 1997; 14 (4): 439-49

[Non-patent document 6] PNAS 2006 103, 7402-7407

[Non-patent document 7] "Seminars in oncology ", (USA), 1993, vol. 20, p. 105-127

[Non-patent document 8] "Genomics", (USA), 2000, vol. 68, p. 348-350

[Non-patent document 9] Oncogene 9: 1567-1574, 1994

[Non-patent document 10] "Cellular and molecular life sciences ", (Switzerland), 2004, vol. 61, p. 2939-2953

[Non-patent document 11] Am J Patol 160: 1487-1494, 2002

[Non-patent document 12] "Journal of cellular physiology ", (USA), 2004, vol. 199, p. 330-358

[Non-patent document 13] "International journal of cancer ", (USA), 2002, vol. 100, p. 49-56

[Non-patent document 14] "Science", (USA), 1994, vol. 263, p. 1281-1284

[Non-patent document 15] "Blood", (USA), 1995, vol. 86, p. 1954-1960

[Non-patent document 16] "Blood", (USA), 2000, vol. 95, p. 3204-3207

[Non-patent document 17] "Blood", (USA), 1999, vol. 94, p. 3265-3268

[Non-patent document 18] "Laboratory investigation; a journal of technical methods and pathology ", (USA), 2003, vol. 83, p. 1255-1265

[Non-patent document 19] "International journal of cancer ", (USA), 2006, vol. 118, p. 1181-1186

[Non-patent document 20] "Blood", (USA), 2006, vol. 107, p. 689-697

[Non-patent document 21] "Blood", (USA), 2006, vol. 107, p. 1617-1623

[Non-patent document 22] "Laboratory investigation; a journal of technical methods and pathology ", (USA), 2005, vol. 85, p. 1544-1554

[Non-patent document 23] "Journal of medicinal chemistry ", (USA), 2006, vol. 49, p. 1006-1015

[Non-patent document 24] J Comb Chem. 8: 401-409, 2006

[Non-patent document 25] "Science", (USA), 1997, vol. 278, p. 1309-1312

[Non-patent document 26] Am J Patol 157: 377-384, 2000

[Non-patent document 27] Blood 90: 2901-2910, 1997

[Non-patent document 28] Am J Patol. 2000 Sea; 156 (3): 781-9.

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Summary of the Invention

The present inventors successfully isolated, from samples obtained from patients with lung cancer, the cDNA and genomic DNA of a novel fusion polynucleotide of a partial EML4 gene fused with a partial ALK gene such as kinase (hereinafter referred to as a polynucleotide of fusion of EML4-ALK v1), which is produced by chromosomal inversion (Examples 1, 2, and 4 (1)). The present inventors also successfully isolated the cDNA and genomic DNA of a novel fusion polynucleotide (hereinafter referred to as a fusion polynucleotide of EML4-ALK v2, fusion polynucleotide of EML4-ALK v3) whose fused regions are different from those of the EML4-ALK v1 fusion polynucleotide (Examples 4 (1), 3 (3), 11 (1) and (7)). Analysis using clinical samples showed that EML4-ALK v1 fusion polynucleotide, EML4-ALK v2 fusion polynucleotide or EML4-ALK v3 fusion polynucleotide is present in some patients with lung cancer (Example 3 and 11 (one)). On the other hand, because the EML4-ALK fusion polynucleotide is an oncogene that presents tumorigenicity depending on its kinase activity (Example 6, 10 (3) and 11 (2)), it was revealed that the fusion polypeptide of EML4-ALK is a tool for screening a therapeutic agent for cancer that is shown to be positive for the fusion polynucleotide. Based on these findings, the present inventors constructed a method to detect the fusion polynucleotide or fusion protein in a sample obtained from a test subject (Examples 3, 4 (2), 5 (2), 9, and 11 (3 )) and, subsequently, a method for screening a fusion polynucleotide inhibitor and / or fusion polypeptide (i.e., a therapeutic agent for cancer shown to be positive for the fusion polynucleotide) causing cancer (Examples 7 , 10 (2), 11 (4), 11 (5) and 11 (8)), and it was confirmed that the compounds obtained by screening have an antitumor effect (Examples 8 (3), 8 (7), 8 (8 ), 11 (6) and 11 (9)). As a result, a test subject in which the fusion polynucleotide has been detected can be treated with cancer using the fusion polynucleotide inhibitor and / or the polypeptide encoded therein. Depending on the detection procedure, the subjects to whom the therapeutic agent can be administered can be selected. As a result, the expected tailor-made medical care can be carried out as a highly effective treatment
using the inhibitor.

Based on these findings, those present inventors disclose in this document a polynucleotide and novel polypeptide useful as screening tools, a screening procedure and a pharmaceutical composition for the Cancer treatment shown to be positive for the gene of fusion of the EML4 gene and the ALK gene. The present inventors unveil additionally a detection procedure useful in detection of the cancer shown to be positive for the fusion gene of EML4 gene and the ALK gene.

Specifically, the present invention will be Refers to: procedures, kits and primer sets as defined in the claims.

None of the aforementioned documents have reported the formation of a fusion gene by means of the EML4 gene and the ALK gene, much less the expression of the fusion gene of the EML4 gene and the ALK gene in some cancer patients. The formation of a fusion gene by means of the EML4 gene and the ALK gene and the expression of this fusion gene in some cancer patients was first found by the present inventors. The screening procedure using a fusion gene of the EML4 gene and the ALK gene is an invention that was first made by the present inventors. In addition, the method for detecting the fusion gene useful in the detection of cancer shown to be positive for the fusion gene, the set of primers useful in this detection and the detection kit are inventions that were provided for the first time. by the findings of the present inventors. Various ALK inhibitors (patent documents 3 to 4 and non-patent documents 20 to 22 and 34) that include 5-chloro- N 4 - [2- (isopropylsulfonyl) phenyl] -N 2 have been reported. 2} - {2-Methoxy-4- [4- (4-methylpiperazin-1-yl) piperidin-1-yl] phenyl} pyrimidine-2,4-diamine and 2 - [(5-bromo-2 - {[ 2-Methoxy-4- (4-methylpiperazin-1-yl) phenyl] amino) pyrimidin-4-yl) amino] -N- methylbenzenesulfonamide. In addition, it has been reported that ALK inhibitors induce growth inhibition and cell death in lymphoma cells that express NPM-ALK fusion proteins (non-patent documents 20 to 22) and inhibit lymphoma (PNAS, January 2, 2007 , 104 (1), 270-275 Epub2006 Dec). However, it is not fully known that these ALK inhibitors have therapeutic applications for cancer (particularly, lung cancer) which is shown to be positive for a fusion gene of the EML4 gene and the ALK gene. The pharmaceutical composition for the treatment of cancer (particularly, lung cancer) that is shown to be positive for a fusion gene of the EML4 gene and the ALK gene is a disclosure that was first provided by the findings of the present inventors.

The polypeptide, polynucleotide, vector expression and cell of the present disclosure can be used in the screening of a substance that inhibits the polypeptide of the present disclosure (particularly, a therapeutic agent for cancer of lung shown to be positive for a gene fusion gene EML4 and the ALK gene). The subjects for whom a fusion gene from EML4 gene and the ALK gene is positive (particularly, patients with lung cancer) can be detected using the presence of polypeptide and / or polynucleotide of the present disclosure as index. According to the screening procedure herein disclosure, a therapeutic agent for cancer can be screened (particularly, a therapeutic agent for lung cancer) that It is shown to be positive for an EML4 gene fusion gene and the ALK gene. The primer and the kit for the detection of the present invention can be used to detect a cancer shown which is positive for a fusion gene of the EML4 gene and the ALK gene. He detection method of the present invention can be used as a procedure to detect cancer (particularly cancer of lung) that is shown to be positive for the fusion gene of EML4 gene and the ALK gene of the present disclosure. Also, according to the detection method of the present invention, may determine whether the therapeutic agent of the present disclosure It may or may not be administered to subjects. The substance that inhibits the Polypeptide of the present disclosure is useful as an agent therapeutic for cancer, particularly lung cancer, which It is shown to be positive for an EML4 gene fusion gene and the ALK gene.

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Brief description of the drawings

Fig. 1. Shows the PCR results. The lane left (46, XX) shows the result when the DNA was used genomic of a normal healthy subject as a substrate, and the right lane (ID # 33) shows the result when the genomic DNA of a cancer patient as a substrate;

Fig. 2. Shows the screening results for EML4-ALK fusion polynucleotide in specimens of patients with lung cancer. The lane "46, XX "shows the result of using peripheral monocytes of a normal healthy female subject, and "ID # 2" to "ID # 42" show the result of using samples obtained from specimens removed from lung cancer patients. In addition, the "NTC" lane Shows the result without added substrate cDNA. The lane "marker" is the lane in which he underwent electrophoresis DNA size marker (upper section). The results of the GAPDH cDNA amplification are shown in the lower section. Sex (M, male; F, female), pathology (S, carcinoma of squamous cells; A, adenocarcinoma; AS, adenoescamoso carcinoma; B, bronchioloalveolar carcinoma) and the presence or absence of EGFR mutation and the presence or absence of smoking history they are shown in the upper part of the figure;

Fig. 3. Shows the tumorigenicity of the genes. The upper section of the figure (3T3) shows cells of 3T3 fibroblasts when a white vector (Vector) was introduced and expression plasmid such as full-length ALK / pMXS (ALK), EML4-ALKV1 / pMXS (EML4-ALK) or EML4-ALK (K589M) / pMXS. The scale bar represents  100 µm. The lower section of the figure (nude mice) shows the result of inoculation of each cell line of 3T3 fibroblasts to nude mice;

Fig. 4. Shows the inhibitory effect of a EML4-ALK fusion polypeptide inhibitor (compound A) on intracellular autophosphorylation. "KM" indicates when cells expressing were used EML4-ALK (K589M) and "EA" shows when they used the BA / F3 cells that express v1. "\ alphap-ALK" (upper panel) shows the immunoblot result when antibody was used phosphorylated anti-ALK and "\ alphaFLAG" (panel bottom) shows the result of the immunoblot when used anti-FLAG antibody;

Fig. 5. Shows the growth potential of cells that express CD8 protein alone (CD8) or coexpress CD8 and ALK (ALK), CD8 and EML4-ALK v1 fusion polypeptide (EA) or CD8 and EML4-ALK (K589M) (KM) in presence (+ IL-3) or absence (-IL-3) of IL-3 The horizontal axis of the figure is the time course (days) and the vertical axis is the number of cells;

Fig. 6 (a). Show dependent change of the time of the number of cells when they were added respective concentrations of compound A to BA / F3 cells that only express CD8 and were grown in the presence of IL-3. (b) Shows the time dependent change of the number of cells when BA / F3 cells expressing v1 were cultured with the respective concentration of compound A in the absence of IL-3 The horizontal axis of the figure is the time course (days) and the vertical axis is the number of cells; Y

Fig. 7. Sample SiRNA-1-siRNA-9.

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Detailed description of the preferred embodiments

Hereinafter, the present invention will be will describe in detail. Gene manipulation techniques described in this document can be practiced according to techniques known in the art such as "Molecular Cloning", Sambrook, J et al., Cold Spring Harbor Laboratory Press, 1989, a unless otherwise specified. Handling techniques of proteins described in this document can be practiced according to techniques known in the art such as "Experimental Protocol on Proteins ", Shujunsha Co. Ltd. 1997, unless it Specify otherwise.

The phrase "the polypeptide of the present disclosure is inhibited "described in this document encompasses both the phrases "the expression of the polypeptide of the present disclosure is inhibited "as" the polypeptide activity of the present disclosure is inhibited. "A" substance that inhibits the polypeptide of the present disclosure "encompasses both a "substance that inhibits the expression of the polypeptide of the present disclosure "as a" substance that inhibits the activity of polypeptide of the present disclosure ".

Polypeptide, polynucleotide, expression vector, cell transformed and procedures to produce the polypeptide from the present disclosure

The polypeptide of the present disclosure includes:

(one)

a polypeptide that is constituted by the sequence of amino acids represented by SEQ ID NO: 2, 7 or 130;

(2)

(to)

a polypeptide comprising the sequence of amino acids represented by SEQ ID NO: 2 and which has an activity of kinase, or a polypeptide comprising a sequence of amino acids derived from the amino acid sequence represented by SEQ ID NO: 2 with the replacement, deletion and / or insertion of 1 to 10 amino acids and that has a kinase activity (named in hereinafter a functionally modified polypeptide equivalent to v1);

(b)

a polypeptide comprising the sequence of amino acids represented by SEQ ID NO: 7 and which has an activity of kinase, or a polypeptide comprising a sequence of amino acids derived from the amino acid sequence represented by SEQ ID NO: 7 with the replacement, deletion and / or insertion of 1 to 10 amino acids and that has a kinase activity (named in hereinafter a functionally modified polypeptide equivalent to v2); Y

(C)

a polypeptide comprising the sequence of amino acids represented by SEQ ID NO: 130 and which has a kinase activity, or a polypeptide comprising a sequence of amino acids derived from the amino acid sequence represented by SEQ ID NO: 130 with the replacement, deletion and / or insertion of 1 to 10 amino acids and that has a kinase activity (named in hereinafter a functionally modified polypeptide equivalent to v3);

(hereinafter, the modified polypeptide functionally equivalent to v1, the modified polypeptide functionally equivalent to v2 and the modified polypeptide functionally equivalent to v3 are collectively referred to as successively a functionally equivalent modified polypeptide); Y

(3)

(to)

a polypeptide comprising a sequence of amino acids with 90% or more identity with the sequence of amino acids represented by SEQ ID NO: 2 and which has an activity kinase (hereinafter referred to as a homologous polypeptide to v1);

(b)

a polypeptide comprising a sequence of amino acids with 90% or more identity with the sequence of amino acids represented by SEQ ID NO: 7 and which has an activity kinase (hereinafter referred to as a v2 homologous polypeptide); Y

(C)

a polypeptide comprising a sequence of amino acids with 90% or more identity with the sequence of amino acids represented by SEQ ID NO: 130 and which has a kinase activity (hereinafter referred to as a polypeptide homologue to v3);

(hereinafter v1 homologous polypeptide, the v2 homologous polypeptide and v3 homologous polypeptide are collectively referred to as a homologous polypeptide).

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\hbox{actividad de
cinasa}

".The "functionally equivalent modified polypeptide" is preferably the "polypeptide comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 with substitution, deletion and / or insertion from 1 to 10 , preferably 1 to several, more preferably 1 to 7, most preferably 1 to 5 amino acids and having a kinase activity. " More preferably, the polypeptide is the "polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and having a

 \ hbox {activity of
kinase 

".

The "homologous polypeptide" is a "polypeptide comprising an amino acid sequence with 90%  or more identity with the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and that has a kinase activity ". Preferably, the homologous polypeptide has a sequence of amino acids with 95% or more, more preferably 98% or more of identity to her.

The "identity" described in this document means an identity value obtained by the search program NEEDLE (J Mol Biol 1970; 48: 443-453) using parameters available by default. The parameters are from following mode:

Penalty per hole = 10

Extension penalty = 0.5

Matrix = EDNAFULL.

The phrase "which has an activity of kinase "means that it has an activity like an enzyme that phosphorylate tyrosine. If a certain polypeptide "has an activity of kinase "is confirmed by an example procedure 7 (2). More preferably, the modified polypeptide functionally equivalent and the homologous polypeptide have combined kinase and tumorigenicity activity. Yes a true polypeptide "has a tumorigenicity" is confirmed by a procedure of Example 6 (1).

Up to this point the polypeptide has been described. of the present disclosure. Hereinafter, the polypeptide that is consisting of the amino acid sequence represented by SEQ ID NO: 2, 7 or 130, the functionally modified polypeptide equivalent and the homologous polypeptide are collectively referred to as successively a "polypeptide of the present disclosure". Of the polypeptides of the present disclosure, the polypeptide that is consisting of the amino acid sequence represented by SEQ ID NO: 2, the functionally modified polypeptide equivalent to v1 and the v1 homologous polypeptide is referred to collectively hereinafter a "type of v1 polypeptide of the present disclosure". He polypeptide that is constituted by the amino acid sequence represented by SEQ ID NO: 7, the modified polypeptide functionally equivalent to v2 and the homologous polypeptide to v2 is collectively referred to as a "v2 polypeptide type of the present disclosure. "The polypeptide that is constituted by the amino acid sequence represented by SEQ ID NO: 130, the Functionally modified polypeptide equivalent to v3 and the v3 homologous polypeptide are referred to collectively hereafter a "type of v3 polypeptide of the present disclosure". A protein like the polypeptide that is made up of the sequence of amino acids represented by SEQ ID NO: 2 is referred to as successive an "EML4-ALK fusion polypeptide v1 ". A protein such as the polypeptide that is constituted by the amino acid sequence represented by SEQ ID NO: 7 is called hereinafter a "fusion polypeptide of EML4-ALK v2 ". A protein like the polypeptide which is constituted by the amino acid sequence represented by SEQ ID NO: 130 is hereinafter referred to as a "polypeptide of fusion of EML4-ALK v3 ". The" polypeptide of fusion of EML4-ALK v1 ", the" polypeptide of fusion of EML4-ALK v2 "and the" polypeptide of fusion of EML4-ALK v3 "are referred to together hereinafter a "fusion polypeptide of EML4-ALK ".

The polypeptide of the present disclosure is, preferably, the "polypeptide comprising the sequence of amino acids represented by SEQ ID NO: 2, 7 or 130 and which has a kinase activity ", more preferably the" polypeptide that comprises the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and which has a kinase and tumorigenicity activity ", most preferably the "polypeptide that is constituted by The amino acid sequence represented by SEQ ID NO: 2, 7 or 130 ".

A polynucleotide encoding the polypeptide of the present disclosure (hereinafter referred to as a "polynucleotide of the present disclosure") is a polynucleotide represented by a nucleotide sequence that encodes the EML4-ALK fusion polypeptide, the Functionally equivalent modified polypeptide or polypeptide counterpart. The polynucleotide of the present disclosure is, preferably, a polynucleotide that is constituted by a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2, 7 or 130, more preferably a polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 1 (particularly preferably, the positions 271 to 3447 in SEQ ID NO: 1), 6 or 129.

Of the polynucleotides of the present disclosure, a gene that encodes the v1 polypeptide type of the This invention is hereinafter referred to as a "type of polynucleotide v1 of the present disclosure. "A gene that encodes the type of v2 polypeptide of the present disclosure be hereafter referred to as a "v2 polynucleotide type of the present disclosure ". A gene encoding the type of polypeptide v3 of this disclosure is hereinafter referred to as a "type of polynucleotide v3 of the present disclosure ".

A "fusion gene of the EML4 gene and the gene ALK "described herein refers to the polynucleotide of This disclosure. A gene that encodes the polypeptide of fusion of EML4-ALK v1, which is a type of polynucleotide v1 of the present disclosure is referred to as successive an "EML4-ALK fusion polynucleotide v1 ". A gene encoding the fusion polypeptide of EML4-ALK v2, which is a type of v2 polynucleotide of This disclosure is hereinafter referred to as a "EML4-ALK v2 fusion polynucleotide". A gene encoding the fusion polypeptide of EML4-ALK v3, which is a type of v3 polynucleotide of This disclosure is hereinafter referred to as a "EML4-ALK v3 fusion polynucleotide". He EML4-ALK v1 fusion polynucleotide, the EML4-ALK v2 fusion polynucleotide and the EML4-ALK v3 fusion polynucleotide are called collectively hereafter a "fusion polynucleotide of EML4-ALK ".

The phrase "cancer shown to be positive for a fusion gene of the EML4 gene and the ALK gene " described in this document means positive cancer for the polynucleotide of the present disclosure (i.e., the polynucleotide of the present disclosure is present) and, preferably means cancer positive for the polynucleotide of EML4-ALK fusion (i.e. cancer positive for EML4-ALK fusion polynucleotide) (i.e. EML4-ALK fusion polynucleotide is Present).

The procedures to produce the polynucleotide of the present disclosure include, but is not they particularly limit to, (1) a procedure that uses the reaction polymerase chain (PCR), (2) a procedure that uses a standard genetic engineering approach (i.e. a method comprising selecting a transformed strain that comprises the desired amino acid sequence of transformed strains  with a cDNA library) and (3) a synthesis procedure chemistry. Each procedure can be practiced in the same way as in WO 01/34785. However, the "novel protein of the present invention "described herein may be interpreted as a protein that is constituted by the polypeptide of the present disclosure described herein, and the "gene of the present invention" described herein can be interpreted as the polynucleotide of the present disclosure described in this document.

In the procedure that uses PCR, the polynucleotide of the present disclosure can be produced, by example, according to procedures described in 1) Procedure of protein gene production, a) First procedure of production in "Embodiments of the Invention" of the document of patent. The mRNA is extracted from cells or tissues that have the ability to produce the protein of the present disclosure, by example, of lung tissues derived from a human patient with lung cancer. Subsequently, this mRNA can undergo reverse transcriptase reaction in the presence of primers at Random or oligo dT primers to synthesize single stranded cDNA. He Single-stranded cDNA obtained can be subjected to PCR using 2 primers that interpose a partial region of the gene of interest to obtain the polynucleotide of the present disclosure or a part thereof. More specifically, the polynucleotide of the present disclosure it can be produced, for example, by a described procedure in Example 4 (1), 11 (1) or 11 (7).

Alternatively, the polynucleotide of the This disclosure can be produced by artificially synthesizing the polynucleotide of the present disclosure as fragments separated by reverse transcription (RT) -PCR and then merging these fragments obtained. For example, (a) 1489 bases located from exon 1 to exon 13 in EML4 (for the type of v1 polynucleotide of the present invention) or 2242 localized bases from exon 1 to exon 20 in EML4 (for polynucleotide type v2 of the present invention) are amplified by RT-PCR using mRNA extracted from cells (e.g., cells) HeLa) or tissues that express endogenously EML4 and using 2 primers that interpose the gene region of interest. For other part, for example, (b) 1691 bases located from exon 21 to exon 30 in ALK (for both the v1 polynucleotide type of the present disclosure as the v2 polynucleotide type of the present disclosure) are amplified by RT-PCR using as mRNA template extracted from cells (for example, Rh30 cells or U-87MG) or tissues that endogenously express ALK and using 2 primers that interpose the gene region of interest. The amplified PCR products of (a) and (b) can be fused to Obtain the polynucleotide of the present disclosure. This fusion can be practiced by devising the primers used in the RT-PCR of (a) and (b). For example, a primer is created adding approximately 10 bases of the nucleotide sequence antisense of the 5 'end of exon 21 in ALK to the 5' end of a antisense primer for RT-PCR amplification of the fragment of (a), and this created primer is used as a primer antisense to amplify the fragment of (a). In addition, a primer it is created by adding approximately 10 bases of the sequence of sense nucleotides of the 3 'end of exon 13 in EML4 (for type of polynucleotide v1 of the present disclosure) or about 10 bases of the 3 'end sense nucleotide sequence of the exon 20 in EML4 (for the type of polynucleotide v2 of the present disclosure) to the 5 'end of a sense primer for the RT-PCR amplification of the fragment of (b), and this created primer is used as a sense primer to amplify the fragment of (b). The polynucleotide of the present disclosure may Obtained by PCR using these 2 types of products as templates PCR obtained and using a sense primer comprising the codon of EML4 initiation and an antisense primer comprising the codon ALK termination. Alternatively, the polynucleotide of the This disclosure can also be obtained through reactions of hybridization and extension using only these 2 types of products PCR obtained without using the sense primer comprising the codon of EML4 initiation and the antisense primer comprising the codon ALK termination.

The expression vector of the present disclosure, the transformed cell of the present disclosure and the procedure to produce the polypeptide of the present disclosure may be practiced, for example, according to procedures described in 2) Procedures for the production of the vector of the invention, the host cell of the invention and the protein recombinant of the invention in "Mode for carrying out the invention "of WO 01/34785. The isolated polynucleotide of the present disclosure can be incorporated back into the DNA of appropriate vector to thus transform a eukaryotic host cell or prokaryotic with him. Alternatively, an appropriate promoter and a sequence that participates in phenotypic expression can enter the vector to make each host cell transformed with it express the polynucleotide.

The expression vector of the present disclosure is not particularly limited as long as you understand the polynucleotide of the present disclosure and express the polypeptide of the present disclosure. Examples thereof they can include an expression vector obtained by inserting the polynucleotide of the present disclosure in an expression vector known in the art, which is appropriately selected according to a host cell used.

Also, the cell of the present disclosure is not particularly limited as long as you understand the polynucleotide of the present disclosure as a result of the nucleic acid transfer by transfection or infection with the expression vector of the present disclosure. For example, the cell of the present disclosure is a host cell that comprises the polynucleotide of the present incorporated disclosure on the chromosome or is it a cell that comprises the expression vector which comprises the polynucleotide of the present disclosure. The cell of the present disclosure is obtained, for example, transfecting or infecting a desired cell with the vector of expression of the present disclosure. More specifically, by example, the expression vector comprising the polynucleotide of the present disclosure and a plasmid to encapsidate (for example, pGP or pE-echo) can be introduced into a cell BOSC23 using a commercially available transfection reagent available, lipofectamine, as described in Example 1, for thus produce an expression retrovirus. A BA / F3 cell can get infected with this retrovirus to produce the cell transformed from the present disclosure.

The desired transformed cell thus obtained It can be grown according to a standard procedure. A protein that it is constituted by the polypeptide of the present disclosure produced by this crop. A medium used in the culture can appropriately selected according to a used host cell from A variety of routine means. For example, a RPMI1640 medium supplemented with serum components such as bovine serum Fetal (SBF) is used for the BA / F3 cell.

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Therefore, the polypeptide of the present disclosure produced by the transformed cell can be separated and purified by a variety of operating techniques of separation known in the art using physical properties or Biochemistry of the polypeptide.

The polypeptide of the present disclosure may merge into the frame with a marker sequence and express in order to get confirmation of protein expression, in addition to protein purification.

Primer set of the present invention

The present invention encompasses a set of primers useful in detecting the presence of the polynucleotide of the present invention.

Specifically, the present invention includes:

(one)

a set of primers to detect the polynucleotide of the present disclosure comprising an antisense primer that is consisting of nucleic acid molecules with at least 16 bases Consecutive that hybridize under restrictive conditions (preferably, more restrictive conditions) to i) a type of polynucleotide v1 of the present disclosure (preferably, EML4-ALK v1 fusion polynucleotide, plus preferably a polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 1), a type of v2 polynucleotide of the present disclosure (preferably, EML4-ALK v2 fusion polynucleotide, plus preferably a polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 6), a type of v3 polynucleotide of the present disclosure (preferably, EML4-ALK v3 fusion polynucleotide, plus preferably a polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 129), ii) a polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 4 which is a genomic sequence that comprises the melting point of a polynucleotide belonging to the EML4-ALK v1 fusion polynucleotide, and / or iii) a polynucleotide that is constituted by the sequence of nucleotides represented by SEQ ID NO: 5 which is one of the sequences comprising the melting point of a polynucleotide which belongs to the fusion polynucleotide of EMK4-ALK v2, and a sense primer that is consisting of a nucleic acid molecule with at least 16 Consecutive bases that hybridize under restrictive conditions (preferably, more restrictive conditions) to chains complementary to any of the above i) to iii);

(2)

a set of primers of a sense primer comprising a oligonucleotide with at least all 16 consecutive bases located in the number of bases 1 to 1759 (preferably, base number 271 to 1759) in SEQ ID NO: 1 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least all 16 consecutive bases located in the number of bases 1760 to 3926 (preferably, base number 1760 to 3447) in SEQ ID NO: 1, or a set of primers that is made up of strings complementary to them, in which the separation between selected positions of the sense and antisense primers in SEQ ID NO: 1 is 1 kb or less, or in which the primers felt and antisense give amplification products of 1 kb or less than size;

(3)

a set of primers of a sense primer comprising a oligonucleotide with at least all 16 consecutive bases located at base numbers 1 to 2242 in SEQ ID NO: 6 and a primer antisense comprising an oligonucleotide complementary to a oligonucleotide with at least all 16 consecutive bases located at base numbers 2243 to 3933 in SEQ ID NO: 6, or a set of primers consisting of strings complementary to them, in which the separation between selected positions of the sense and antisense primers in SEQ ID NO: 6 is 1 kb or less, or in which the primers felt and antisense give amplification products of 1 kb or less than size;

(4)

a set of primers of a sense primer comprising a oligonucleotide with at least all 16 consecutive bases located at base numbers 1 to 3629 in SEQ ID NO: 4 and a primer antisense comprising an oligonucleotide complementary to a oligonucleotide with at least all 16 consecutive bases located in the bases number 3630 to 3979 in SEQ ID NO: 4, or a set of primers consisting of strings complementary to them, in which the separation between selected positions of the sense and antisense primers in SEQ ID NO: 4 is 1 kb or less, or in which the primers felt and antisense give amplification products of 1 kb or less than size;

(5)

a set of primers of a sense primer comprising a oligonucleotide with at least all 16 consecutive bases located in the bases number 1 to 579 in SEQ ID NO: 5 and a primer antisense comprising an oligonucleotide complementary to a oligonucleotide with at least all 16 consecutive bases located in the bases number 580 to 853 in SEQ ID NO: 5, or a set of primers consisting of strings complementary to them; Y

(6)

a set of primers of a sense primer comprising a oligonucleotide with at least all 16 consecutive bases located in the number of bases 1 to 700 in SEQ ID NO: 129 and a antisense primer comprising a complementary oligonucleotide to an oligonucleotide with at least all 16 consecutive bases located in bases no. 701 to 1691 in SEQ ID NO: 129, or a set of primers consisting of strings complementary to them, in which the primers felt and antisense give amplification products of 1 kb or less than size. Examples of a preferable primer set include:

(7)

he set of primers or set of primers that is constituted by complementary chains of the same according any of (3) to (6), in which the primers felt and antisense are represented by (i) SEQ ID NO: 8 and 9, (ii) SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, or 33 and 34, (iii) SEQ ID NO: 35 and 36, 37 and 38, 39 and 18, 41 and 20, 43 and 22, 45 and 24, 47 and 26, 49 and 28, 51 and 52, 53 and 54, or 55 and 34, (iv) SEQ ID NO: 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, or 79 and 80, (v) SEQ ID NO: 81 and 62, 83 and 84, 85 and 68, 87 and 88, 89 and 70, 91 and 92, 93 and 74, 95 and 96, 97 and 98, or 99 and 100, or (vi) SEQ ID NO: 131 and 82, 132 and 62, 133 and 64, 134 and 66, 135 and 68, 136 and 70, 137 and 72, 138 and 74, 139 and 76, 140 and 78, or 141 and 80.

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The primer sets (7) (i) (in the Example 3 (1)), (7) (ii) (in Example 3 (2)), (7) (iii) (in Example 3 (3)), and (7) (iv) and (v) (in Example 4 (2)), and (7) (vi) (in Example 11 (3)) were used to detect the presence of the polynucleotide of the present divulgation.

Melting point in this specification means a point at which a part derived from the gene is fused EML4 and a part derived from the ALK gene. The melting point in SEQ ID No: 1 is the point at which a position nucleotide is fused base 1759 and a nucleotide base position 1760. The point of merge in SEQ ID No: 6 is the point at which a merge is merged base position nucleotide 2242 and a position nucleotide of base 2243. The melting point in SEQ ID No: 129 is the point in the that a base position nucleotide 700 and a core position nucleotide 701. The melting point in SEQ ID No: 4 is the point at which a position nucleotide is fused from base 3629 and a base position nucleotide 3630. The point of merge in SEQ ID No: 5 is the point at which a merge is merged base position nucleotide 579 and a position nucleotide of base 580.

The "restrictive conditions" in this Descriptive report include hybridization conditions in the order of "5 x SSPE, 5 \ dissolution of Denhardt, SDS at 0.5%, 50% formamide, 200 µg / ml salmon sperm DNA, 42 ° C overnight "and washing conditions in the order of "0.5 x SSC, 0.1% SDS, 42 ° C". The "most conditions restrictive "include hybridization conditions in the order of "5 x SSPE, 5 x Denhardt solution, 0.5% SDS, 50% formamide, 200 µg / ml salmon sperm DNA, 42 ° C overnight "and washing conditions in the order of" 0.2 x SSC, 0.1% SDS, 65 ° C. "

The primer sets of the present invention can be used as primer sets for amplify and detect the polynucleotide of the present disclosure. For the use of primers, their chain length is normally of 15 to 40 bases, preferably 16 to 24 bases, more preferably from 18 to 24 bases, particularly preferably from 20 to 24 bases.

The procedures for producing primers of the The present invention is not particularly limited. The primers of the present invention can be produced by the chemical synthesis procedure used for the procedure for produce the polynucleotide of the present disclosure.

Detection procedure and kit for the detection of present invention

The present invention encompasses a procedure to detect the polynucleotide of the present disclosure and a procedure to detect a fusion protein that is constituted by the polypeptide of the present disclosure. Specifically, an aspect that Understand the stage. Specifically, the procedure to detect The polynucleotide of the present disclosure comprises the step from

(one)

detect the presence of polynucleotide of the present disclosure in a sample obtained from a test subject

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A sample collected from a test subject (a sample separated from the body), specifically any fluid collected body (preferably blood), washing fluid bronchoalveolar, biopsy sample or sputum sample, is used as the sample obtained from a test subject. It is preferably used a biopsy sample of the affected part of the subject's lung test or a sputum sample of the test subject. May genomic DNA extracted from the sample or a product of transcription (product as a result of transcription and genome translation; for example mRNA, cDNA, or a protein) of same. Particularly preferably, for use, mRNA or CDNA

In the procedure to detect the polynucleotide of the present disclosure, the "step of detecting the presence of the polynucleotide "can be practiced by detecting the presence of the polynucleotide represented by SEQ ID NO: 4 (genomic sequence comprising the melting point) or SEQ ID NO: 5 (genomic sequence comprising the melting point) in the genome of the sample obtained from a test subject or by detecting the presence of mRNA or DNA corresponding to the type of polynucleotide v1 of the present disclosure (preferably, the polynucleotide of fusion of EML4-ALK v1), the type of polynucleotide v2 of the present disclosure (preferably, the polynucleotide of fusion of EML4-ALK v2) or the type of v3 polynucleotide of the present disclosure (preferably, the EML4-ALK v3 fusion polynucleotide) preparing a transcription product (eg mRNA or cDNA) of DNA genomic extracted from the sample obtained from a test subject.

Genomic DNA extraction can be performed by a procedure known in the art and can be performed conveniently with a commercially available DNA extraction kit available.

The detection stage in stage (1) can be practiced according to a gene analysis procedure known in the technique (for example, PCR commonly used as a procedure for gene detection, CSF (ligase chain reaction), SDA (chain shift amplification), NASBA (amplification based on nucleic acid sequences), ICAN (amplification isothermal and chimeric nucleic acid initiated by primers), LAMP (loop-mediated isothermal amplification), TMA (TMA system of gene probes) and well-known procedures such as a microarray). For example, a hybridization technique is used using as a probe a nucleic acid that hybridizes with the polynucleotide of the present invention or a technique of gene amplification using as primers DNA that hybridizes with the polynucleotide of the present invention. Specifically for the measurement uses a nucleic acid, for example, mRNA, derived from the sample obtained from a test subject. MRNA level is measured by a gene amplification reaction procedure that use primers designed to specifically amplify the polynucleotide sequence of the present disclosure. The primers used in the detection method herein invention or the primers contained in the kit for the detection of the present invention are not particularly limited while specifically amplify the polynucleotide sequence of the present invention These primers are designed based on the nucleotide sequence of the polynucleotide of the present invention. The design of primers for a procedure of PCR amplification monitoring can be achieved using Primer Express primer design software (PE Biosystems). PCR products with a large size reduce the efficiency of amplification. Therefore, it is appropriate that the primers felt and antisense can be designed to give amplification products of 1 kb or less in size in the amplification of mRNA or cDNA. Plus specifically, a sense primer (5 'primer) and a primer antisense (3 'primer) are designed from a part that encodes EML4 (for example, any part within the region of EML4 gene of the EML4-ALK fusion polynucleotide (particularly cDNA)) and a part encoding ALK (by example, any part within the ALK gene region of the EML4-ALK fusion polynucleotide (particularly cDNA)), respectively. They are preferably used the primers contained in the kit for the detection of this invention, more preferably, the most suitable primers contained in the kit for the detection of the present invention. Whether the gene of interest (the complete sequence or its part specific) is amplified as not, can be confirmed by Proper procedure for each amplification technique. By For example, for PCR, PCR products can be subjected to analysis by agarose gel electrophoresis and bromide staining of ethidium to confirm whether an amplification fragment with the Interest size is obtained or not. If the fragment of amplification with the size of interest, then the polynucleotide of the present invention is present in the sample obtained from a test subject In this way the presence of the polynucleotide of the present disclosure.

Detection using the hybridization technique is performs, for example, by a hybridization of Northern, Dot transfer or DNA microarray procedure. To detect the polynucleotide in hybridization, a probe comprising a nucleic acid molecule with at least 32 Consecutive bases that hybridize under restrictive conditions (preferably, more restrictive conditions) with a polynucleotide of the present disclosure; or chains complementary to it, and comprising positions 1744 a 1775 of the nucleotide sequence represented by SEQ ID NO: 1, positions 2227 to 2258 of the nucleotide sequence represented by SEQ ID NO: 6, positions 685 to 716 of the nucleotide sequence represented by SEQ ID NO: 129, the positions 3614 to 3645 of the nucleotide sequence represented by SEQ ID NO: 4, positions 564 to 595 of the sequence of nucleotides represented by SEQ ID NO: 5; or complementary chains  of the same. In addition, an amplification technique of genes such as RT-PCR. In the procedure of RT-PCR, the presence of the polynucleotide of the This disclosure can be analyzed more quantitatively using a PCR amplification monitoring procedure (PCR in real time) (Genome Res. 6 (10), 986, 1996) in the procedure of gene amplification. For example, ABI PRISM 7900 can be used (PE Biosystems) as a monitoring procedure for PCR amplification. Real time.

PCR is a procedure known in the art and can be conveniently performed using a device commercially available and a kit for real-time PCR.

A procedure to detect a protein from fusion encoded by the polynucleotide of the present disclosure understand the stage of

(2)

detect the presence of the polypeptide of the present disclosure in a sample obtained from a subject of proof.

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Such a detection stage can be practiced. preparing a solubilized solution derived from a sample obtained from a test subject (for example, a tissue or cell cancerous obtained from the test subject) and detecting the polypeptide of the present disclosure (particularly, the EML4-ALK v1 fusion polypeptide contained in the same for an immunological measurement or measurement procedure of enzymatic activity using antibodies anti-EML4 and anti-ALK in combination. Preferably an approach can be used using an antibody monoclonal or polyclonal specific for the polypeptide of the present disclosure (particularly, the fusion polypeptide of EML4-ALK v1) such as enzyme immunoassay, ELISA sandwich type with two antibodies, fluoroimmunoassay, radioimmunoassay or Western blotting.

More preferably, the presence of Polypeptide of the present disclosure can be detected, as shown in Example 9, subjecting cell extracts from a cell that probably has the polypeptide of the present disclosure to immunoprecipitation with an antibody anti-EML4 and performing detection using a anti-ALK antibody for precipitates. At procedure of Example 9 can also be used the immunoprecipitation with the anti-ALK antibody and the detection with the anti-EML4 antibody. So, after performing immunoprecipitation and detection, it prefers to further confirm that the protein detected by the detection antibody has the size of the polypeptide of the Present disclosure of interest. The antibodies used in this detection can be any antibody that can bind specifically to a sequence of polypeptides from exon 1 to exon 20 (preferably, from exon 1 to exon 13; more preferably from exon 1 to exon 6) of EML4 or a sequence of exon polypeptides 21 to exon 30 of ALK, and may be monoclonal antibodies or polyclonal

If the polynucleotide of the present disclosure or the polypeptide of the present disclosure is detected in the sample obtained from a test subject, the test subject is a target (patient) who has cancer that is shown to be positive for the polynucleotide of the present disclosure and serves as a target to which can be administered the pharmaceutical composition of the present disclosure.

The kit for the detection of this invention comprises at least sense and antisense primers designed to specifically amplify the polynucleotide of the present disclosure. The set of primers felt and antisense are a set of polynucleotides that act as primers for polynucleotide amplification of the present divulgation. Examples thereof include sets of primers (1) to (7) described in paragraph <Set of primers of the present invention>. Primer sets Preferable are sets of primers (2) to (7). The sets of more preferable primers are primer sets (7).

Examples of other reagents that may be contained in the kit for the detection of the present invention may include reagents (e.g., Taq polymerase, substrates nucleotide and buffer solutions) necessary for PCR.

Screening procedure of this disclosure

The screening procedure herein disclosure encompasses [1] a procedure to screen a substance which inhibits the polypeptide of the present disclosure and [2] a procedure to screen a therapeutic agent for cancer (preferably, lung cancer) that is shown to be positive for the polynucleotide of the present disclosure.

[1] Procedure for screening a substance that inhibits polypeptide of the present disclosure (which inhibits activity and / or expression of the polypeptide of the present disclosure)

The procedure to screen a substance that inhibits the polypeptide of the present disclosure is not particularly limited while understanding the following stages (i) to (iii):

(i)

put test substances in contact with the polypeptide of the present disclosure or a cell that expresses the polypeptide of the present disclosure;

(ii)

analyze whether the polypeptide is inhibited or not, and

(iii)

select a substance that inhibits the polypeptide

Preferably, the substance that inhibits the Polypeptide of the present disclosure can be screened by procedures described in Examples 7 (3), 7 (4), 8 (3), 8 (5) - 8 (9), 10 (3), 11 (4) - (6), 11 (8), and 11 (9).

The screening procedure [1] of the present disclosure includes the following procedures (a), (b) or (C):

(to)

in vitro screening procedure;

A procedure to screen a substance that inhibits the activity of the polypeptide of the present disclosure comprising the steps of (1) putting test substances in contact with the polypeptide herein disclosure, (2) analyze whether polypeptide activity is inhibited or not and (3) select a substance that inhibits the activity of the polypeptide;

(b)

screening procedure based on cells;

A procedure to screen a substance that inhibits the activity of the polypeptide of the present disclosure comprising the steps of (1) putting test substances in contact with a cell that expresses the polypeptide of the present disclosure, (2) analyze whether the polypeptide activity is inhibited or not, and (3) select a substance that inhibits polypeptide activity; Y

(C)

screening procedure based on the expression inhibition; a procedure to screen a substance that inhibits the expression of the polypeptide of the present disclosure comprising the steps of (1) putting substances of test in contact with a cell that expresses the polypeptide of the Present disclosure, (2) analyze whether polypeptide expression is inhibited or not, and (3) select a substance that inhibits the polypeptide expression.

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Each screening procedure (a) - (c) is will describe later. The cell that expresses the polypeptide of the present disclosure comprises a cell that naturally expresses the polypeptide of the present disclosure (for example NCI-H2228) and a cell that produces the expression of polypeptide of the present disclosure by its transformation with a vector comprising the polynucleotide of the present disclosure. The preferable cell is the cell that produces the expression of the polypeptide of the present disclosure by its transformation with a vector comprising the polynucleotide of the present divulgation.

(a) In vitro screening procedure

The in vitro screening method comprises: putting test substances in contact with the purified polypeptide of the present invention by addition (contact step); analyze whether the activity of the polypeptide of the present disclosure is inhibited or not by the test substance (s) in comparison with the activity of the polypeptide of the present disclosure that has not been contacted with the test substance (step of analysis); and selecting a substance that inhibits the activity of the polypeptide of the present disclosure (ie, a therapeutic agent for cancer, particularly, a therapeutic agent for lung cancer).

In the screening procedure of the present disclosure, each step can be practiced specifically, for example, as described below. The test substances are contacted with the purified polypeptide of the present disclosure by addition. After the addition of ATP, the activity of the polypeptide is measured. The solvents (for example, DMSO) for the test substance are contacted as a control with the purified polypeptide by mixing. After the addition of ATP, the activity of the polypeptide is measured. As a noise control a condition can be set without the addition of ATP. It is analyzed whether the activity of the polypeptide of the present disclosure is inhibited or not by the test substance (s). Whether the activity (i.e., phosphorylation activity) of the polypeptide of the present disclosure is inhibited or not by the test substance (s) can be determined by analyzing a change induced by the test substance at the phosphorylation level of Tyrosine of the polypeptide of the present disclosure. Specifically, when the addition (i.e., contact) of a test substance inhibits the activity (i.e., phosphorylation activity) of the polypeptide of the present disclosure compared to the addition (i.e., contact) of the control of solvent, this test substance is selected as a substance that inhibits the activity of the polypeptide of the present disclosure, (i.e., a therapeutic agent for cancer, particularly, a therapeutic agent for lung cancer). The in vitro screening process of the present disclosure comprises a screening procedure carried out in a similar manner to the previous one, except that in the previous stages the peptide substrate is added and mixed before the addition of ATP, and the activity of the Polypeptide of the present disclosure is determined by analyzing the phosphorylation activity in the peptide substrate by the polypeptide of the present disclosure (i.e., analyzing a change in the level of phosphorylation in the peptide substrate by the polypeptide of the present disclosure in order to determine whether a test substance inhibits the activity of the polypeptide of the present disclosure). Of the screening methods of the present disclosure, the in vitro screening procedure is preferably practiced under the conditions described in Example 7 (3) or 11 (4). A substance that can inhibit 50% or more of activity by this procedure at a concentration of 10 µM or less, preferably 1 µM or less, more preferably 0.1 µM or less, is selected as a substance that inhibits the Polypeptide activity of the present disclosure.

(b) Cell based screening procedure

The cell-based screening procedure comprises: putting test substances in contact with a cell expressing the polypeptide of the present disclosure by mixture (i.e. addition) (contact stage); analyze if the Polypeptide activity of the present disclosure is inhibited or not by the test substance (s) compared to the activity of the polypeptide of the present disclosure that is not has contacted the test substance (stage of analysis); and select a substance that inhibits the activity of polypeptide of the present disclosure (ie, an agent therapeutic for cancer, particularly a therapeutic agent for lung cancer). This screening procedure can be specifically practiced, for example, as described in continuation.

First, the test substances or controls of solvent (for example, DMSO) is contacted with a cell expressing the polypeptide of the present disclosure. The cells They are grown for a given time. The activity (that is, the autophosphorylation activity) of the polypeptide of the present disclosure is measured using cell lysates prepared from of cells cultured by dissolution, by SDS electrophoresis known in the art and immunoblot using an antibody phosphorylated anti-ALK (for example, technology cell signaling) to analyze whether the activity of the polypeptide of the present disclosure is inhibited or not by the test substance (s). If the activity of polypeptide of the present disclosure is inhibited or not by the test substance (s) can be determined analyzing a change induced by the test substance in the tyrosine phosphorylation level (i.e. autophosphorylation) of polypeptide of the present disclosure. Specifically, when the addition (i.e. contact) of a test substance inhibits the activity of the polypeptide of the present disclosure in comparison with the addition (i.e. contact) of the control of solvent, this test substance is selected as a substance that inhibits the activity of the polypeptide of the present disclosure (i.e. a therapeutic agent for cancer, particularly, a therapeutic agent for lung cancer). From the screening procedures of this disclosure, preferably, the cell-based screening procedure will be practice under the conditions described in Example 7 (4), 10 (3), 11 (5) or 11 (8). One is selected substance that can inhibit 50% or more activity by this procedure at a concentration of 10 µM or less, preferably 1 µM or less, more preferably 0.1 µM or lower

(c) Screening procedure based on the inhibition of The expression

The screening procedure based on the expression inhibition comprises: putting test substances in contact with a cell expressing the polypeptide of the present Disclosure by mixing (i.e. addition) (stage of Contact); analyze whether the expression of the polypeptide of the present disclosure is inhibited or not by the substance (s) of test compared to polypeptide expression of the present disclosure that has not contacted the test substance (analysis stage); and select one substance that inhibits the expression of the polypeptide of the present disclosure (i.e. a therapeutic agent for cancer, particularly, a therapeutic agent for lung cancer, which shows that it is positive for the polynucleotide of the present divulgation). This screening procedure can be practiced specifically, for example, as described below.

Test substances or controls solvent (for example, DMSO) is contacted with any cell expressing the polypeptide of the present disclosure. After cultivation, the extracts are prepared from the cells and subsequently used to analyze whether the expression of polypeptide of the present disclosure is inhibited or not by the test substance (s). If the expression of polypeptide of the present disclosure is inhibited or unable analyzed by analyzing whether mRNA or protein expression of the Polypeptide of the present disclosure is inhibited or not. Plus specifically, the mRNA or protein level of the polypeptide of The present disclosure present in cell extracts is identified by an expression level analysis procedure known in the art, for example, Northern blot, PCR quantitative, immunoblot or ELISA. More specifically, the mRNA inhibition or protein expression of the polypeptide of The present disclosure can be analyzed by a procedure described in Example 8 (5) or 8 (6). If the expression of the polypeptide of the present disclosure is inhibited or not by the test substance (s) can be determined analyzing a change induced by the test substance at the level of expression of the polypeptide of the present disclosure. Specifically, when the contact of a test substance inhibits the level of expression (i.e. mRNA level or proteins) of the polypeptide of the present disclosure in comparison with the contact of the solvent control, this test substance is selected as a substance that inhibits the expression of polypeptide of the present disclosure (ie, an agent therapeutic for cancer, particularly a therapeutic agent for lung cancer). Of the screening procedures of the present disclosure, preferably, the screening procedure based on inhibition of expression is practiced in the conditions described in Example 8 (5) or 8 (6). Be select a substance that can inhibit 50% or more activity  by this procedure at a concentration of 10 µM or less, preferably 1 µM or less, more preferably 0.1 µM or lower Preferably, the test substance selected It has an inhibitory activity in all the cells used. Without However, a test substance can also be selected that It has an inhibitory activity in a cell.

[2] Procedure to screen a therapeutic agent for cancer shown to be positive for the polynucleotide of the present disclosure

It was revealed that the fusion polynucleotide of EML4-ALK is an oncogene (Example 6 and 11 (2)) and the presence of the EML4-ALK fusion polynucleotide It was detected in some patients with lung cancer. Additionally, it was revealed that independent cell growth of the anchor was inhibited (i.e. an effect is shown anticancer) by inhibition of activity and / or expression of polypeptide of the present disclosure (Example 8, 11). It showed itself that the substance that inhibits the polypeptide of the present disclosure (which inhibits the activity and / or expression of the polypeptide of the present disclosure) has a therapeutic effect on the Cancer. Specifically, the procedure to screen a substance which inhibits the polypeptide of the present disclosure (which inhibits the activity and / or expression of the polypeptide of the present disclosure) mentioned above [1] can be used as a procedure to screen a therapeutic agent for cancer (preferably, lung cancer) shown to be positive for polynucleotide of the present disclosure.

The screening procedure [2] of the present invention further comprises, in addition to analyzing whether the polypeptide of the present disclosure is inhibited or not and select a substance that inhibits the polypeptide of the present disclosure, the step of confirming that the selected test substance has a therapeutic activity against cancer (particularly cancer of lung) shown to be positive for the polynucleotide of This disclosure.

The stage of confirming that the test substance selected has a therapeutic activity against cancer

Examples of the stage of confirming that the Selected substance has a therapeutic activity against the cancer (particularly, lung cancer) that is shown to be Positive for the polynucleotide of the present disclosure include a stage of practicing an evaluation procedure known in the technique or a modified procedure thereof, for example, a procedure comprising analyzing the therapeutic activity of the selected substance against cancer (particularly, cancer of lung) dealing with the substance cultured cells or animals of tumor model expressing the polypeptide of the present disclosure (Clinical Oncology, 2nd ed., Cancer and Chemotherapy Publishers, Inc.).

The stage of confirming that the test substance selected has a therapeutic activity against cancer (particularly, lung cancer) that is shown to be positive for the polynucleotide of the present disclosure is, preferably, the step of confirming that the test substance selected has the growth inhibitory effect and / or inducing effect of cell death in cells using cells cancer-derived cancers (particularly those derived from lung cancer) that endogenously express the polypeptide of the present disclosure (for example, NCI-H2228), the stage of confirming that the selected test substance has the inhibitory effect on the independent growth of the anchorage of cells transformed by the expression of the polypeptide of the present disclosure and / or the stage of confirming that the substance of Selected test has the inhibitory effect on growth of tumor formed in nude mouse inoculated with a cell that expresses the polynucleotide of the present disclosure.

A model carrier animal can be used for cancer that serves as a tumor model animal in which cells cancerous endogenously expressing the polypeptide of the present disclosure (for example, NCI-H2228) or cells transformed by the expression of the polypeptide of the present disclosure are transplanted subcutaneously, intradermally or intraperitoneally or to each organ (for example, a naked mouse to which the NIH3T3 cells that produce the expression are transplanted of the polypeptide of the present invention). In addition, you can also an animal that produces the polynucleotide expression of EML4-ALK fusion.

Examples of test substances used in the Screening procedure of the present disclosure may include, but not particularly limited to, commercially compounded available (including peptides), a variety of compounds (including peptides) known in the art and recorded in chemical files, groups of compounds obtained by a technique of combinatorial chemistry (N. Terrett et al., Drug Discov. Today, 4 (1): 41, 1999), culture supernatants of microorganisms, natural components derived from plant or marine organisms, animal tissue extracts, double stranded nucleic acids, antibodies or antibody fragments and compounds (including peptides) chemically or biologically modified from compounds (including peptides) selected by the procedure of screening of the present disclosure.

Use for the preparation of pharmaceutical composition for Cancer treatment shown to be positive for the polynucleotide of the present disclosure, and nucleic acid double stranded

The present disclosure encompasses a pharmaceutical composition for the treatment of cancer that is shown to be positive for the polynucleotide of the present disclosure comprising as active ingredient a substance obtained by the screening method of the present disclosure. The present disclosure encompasses the use of a substance that inhibits the polypeptide of the present disclosure (eg, a substance obtained by the screening process of the present disclosure [eg, a double stranded nucleic acid (including siRNA), protein (including a antibody or antibody fragment), peptide or other compounds]) for the preparation of a pharmaceutical composition
Therapeutics for the treatment of cancer shown to be positive for the polynucleotide of the present disclosure.

The active ingredient in the pharmaceutical composition can be selected by the screening method of the present disclosure. Examples of the substance selected by the screening process of the present disclosure may include compounds A to D and a double stranded nucleic acid described in Examples 7 and 8 below, respectively. Alternatively, a compound selected by the screening method of the present disclosure of a low molecular weight compound with an inhibitory activity against ALK (ALK inhibitor) known in the art can be used as an active ingredient in the pharmaceutical composition. The ALK inhibitor can be exemplified by ALK inhibitors described in WO 2005/097765 and WO 2005/016894 (particularly, 2,4-pyrimidinediamine derivatives). In particular, a compound described in Wan W et al., Blood 107: 1617-1623, 2006, in addition to WHI-P131 (4- (4'-hydroxyphenyl) amino-6,7-dimethoxyquinazoline) and WHI-P154 (4 - [(3'-Bromo-4'-hydroxyphenyl) amino] -6,7-dimethoxyquinazoline); hereinafter referred to as a compound A; Marzec M et al., Lab Invest 85: 1544-1554, 2005) (both, EMD Biosciences). Alternatively, N - [2- (3-chlorophenyl) ethyl] -2 - [({[4- (trifluoromethoxy) phenoxy] acetyl} amino) methyl] -1,3-thiazol-4-carboxamide can be used as an ALK inhibitor (WO 2005/097765; hereinafter referred to as a compound B), 5-chloro- N 4 - [2- (isopropylsulfonyl) phenyl] - N 2 - {2-methoxy-4- [4 - (4-methylpiperazin-1-yl) piperidin-1-yl] phenyl} pyrimidine-2,4-diamine (WO 2005/016894; hereinafter referred to as a compound C) or 2 - [(5-bromo-2 - {[2-Methoxy-4- (4-methylpiperazin-1-yl) phenyl] amino} pyrimidin-4-yl) amino] -N- methylbenzenesulfonamide (WO 2005/016894; hereinafter referred to as a compound D).

The double stranded nucleic acid exemplified as active ingredient in the pharmaceutical composition comprises a part of double stranded nucleic acid (RNA or DNA) and, preferably, protuberant nucleotides of the 3 'end of the chains of transcription and complementary and induces iRNA. IRNA is a phenomenon evolutionarily conserved that is produced by a nucleic acid double stranded with 21 to 23 bases produced by the RNase endonuclease III (Genes Dev. 15, 485-490, 2001). Nucleotides 3 'end protuberants are respectively any acid nucleic with 1 or 2 bases, preferably 2 bases. The number of bases (21 to 23 bases) described above is the number of bases of the transcription or complementary chain including its protuberant nucleotide The transcription chains and complementary can have the same number of bases or a number of different bases and preferably have the same number of bases.

For example, U (uridine), A (adenosine), G (guanosine) or C (cytidine) can be used as ribonucleic acids which constitute the protuberant nucleotides of the 3 'end of the double stranded nucleic acid. For example, dT (deoxythymidine), dA (deoxyadenosine), dG (deoxyguanosine) or dC (deoxycytidine) can used as deoxyribonucleic acids that constitute the protuberant nucleotides of the 3 'end thereof.

The double stranded nucleic acid that can be used as active ingredient in the pharmaceutical composition comprises a double-stranded part designed based on the bases in the positions 1743 to 1761, 1744 to 1762, 1750 to 1768, 1753 to 1771, 1756 to 1774, or 1757 to 1775 in SEQ ID NO: 1 and has an inhibitory activity on the expression of the polypeptide of the present disclosure (termed hereinafter a double stranded nucleic acid of the present divulgation). Examples of preferable aspects of a nucleic acid such double stranded include the siRNA-1 to SiRNA-6 described in example 8 (i.e. a double stranded nucleic acid in which a chain is constituted by the nucleotide sequence represented by SEQ ID NO: 111 and the another chain is constituted by the nucleotide sequence represented by SEQ ID NO: 112; a double stranded nucleic acid in the that a chain is constituted by the nucleotide sequence represented by SEQ ID NO: 113 and the other chain is constituted by the nucleotide sequence represented by SEQ ID NO: 114; a double stranded nucleic acid in which a chain is constituted by the nucleotide sequence represented by SEQ ID NO: 115 and the  another chain is constituted by the nucleotide sequence represented by SEQ ID NO: 116; a double stranded nucleic acid in which a chain is constituted by the nucleotide sequence represented by SEQ ID NO: 117 and the other chain is constituted by the nucleotide sequence represented by SEQ ID NO: 118; a double stranded nucleic acid in which a chain is constituted by the nucleotide sequence represented by SEQ ID NO: 119 and the another chain is constituted by the nucleotide sequence represented by SEQ ID NO: 120; and a double stranded nucleic acid in which a chain is constituted by the nucleotide sequence represented by SEQ ID NO: 121 and the other chain is constituted by the nucleotide sequence represented by SEQ ID NO: 122). The double stranded nucleic acid of the present invention can be produced by standard procedures (for example, J. Am. Chem. Soc. 120, 11820-11821, 1998; and Methods, 23, 206-217, 2001). Alternatively, one manufacturer per double-stranded nucleic acid contract (for example, RNAi Co. Ltd.) is well known to those skilled in the art and can be used in the production of double stranded nucleic acid. Be confirmed by an ARNip sequence design system (version commercial siDirect (registered trademark), RNAi Co. Ltd.) that the target sequences of siRNAs-1 to SiRNA-6 were specific for the polypeptide of the present disclosure.

The double stranded nucleic acid of the present invention can be designed based on nucleotide sequences of DNA (bases at positions 1743 to 1761, 1744 to 1762, 1750 to 1768, 1753 to 1771, 1756 to 1774, or 1757 to 1775 in SEQ ID NO: 1) elected as a target for siRNA-1 to SiRNA-6. Such a double stranded nucleic acid inhibits the polypeptide of the present disclosure, as with the SiRNA-1 to siRNA-6. For example, him ARNip can be designed by having a double-stranded part that is consisting of an RNA nucleotide sequence directly converted from the target DNA nucleotide sequence complete. Alternatively, the chimeric double stranded nucleic acid DNA-RNA (comprising both RNA and DNA in the identical chain) can be designed having an RNA sequence converted from any part of the sequence of target DNA nucleotides. In addition, a nucleic acid can be designed double-stranded hybrid in which one chain is DNA and the other chain is RNA and is produced for use. The sequence conversion of RNA nucleotides from the DNA nucleotide sequence described in this document means that the dT in the sequence of DNA nucleotides are converted to U and other bases, that is, dA, dG and dC become A, G and C, respectively.

The double stranded nucleic acid of the present disclosure presented an inhibitory effect on growth independent of the anchor of a cell that expresses the polypeptide of the present disclosure (Example 8 (7)). Therefore the double stranded nucleic acid of the present disclosure may be used to prepare the pharmaceutical composition for the Cancer treatment shown to be positive for the polynucleotide of the present disclosure.

A therapeutic effect on cancer that is shows that it is positive for the polynucleotide of the present Disclosure can be confirmed by using a procedure generally known to those skilled in the art or a modified procedure (see "The step of confirming that the selected test substance has an activity Therapeutic against cancer ").

A preparation that comprises as a principle active a substance that inhibits the polypeptide of the present disclosure (for example, a substance [for example, an acid double stranded nucleic acid, protein (including an antibody or antibody fragment), peptide or other compounds] obtained by the screening procedure of the present disclosure) may be prepared as a pharmaceutical composition using vehicles, excipients and / or other pharmacologically acceptable additives normally used in the production of the preparation according to the type of the active substance

Examples of administration may include: oral administration using tablets, pills, capsules, granules, fine granules, powders or oral liquid agents; Y parenteral administration using injections for injection intravenous (including intravenous drip), injections intramuscular or subcutaneous injection, suppositories, agents percutaneous administration or transmucosal administration agent. Particularly preferred is parenteral administration such as intravenous injection for peptides that are digested in the stomach.

To prepare a solid composition for oral administration, 1 or more active substances may be mixed with at least one inactive diluent, for example, lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolidone or magnesium aluminometasilicate. The composition may contain additives other than diluent inactive, for example, lubricants, disintegrants, stabilizers or solvents or solubilizers according to a standard procedure. The tablets or pills may be coated, if necessary, with a sugar coating or with a film such as a substance Gastrosoluble or enteric.

A liquid composition for administration by orally it can comprise, for example, an emulsion, solution, suspension, syrup or elixir and may contain an inactive diluent generally used, for example, purified water or ethanol. The composition may contain additives other than diluent inactive, for example, humectants, suspensions, sweeteners, aromas or antiseptics.

A parenteral injection may comprise a aseptic aqueous or non-aqueous solution, suspension or emulsion. A water soluble solution or suspension may contain, for example, distilled water or saline solution for injection as a diluent. A water-insoluble solution or suspension may contain, by example, propylene glycol, polyethylene glycol, vegetable oil (for for example, olive oil), alcohols (for example, ethanol) or polysorbate 80 as diluent. The composition may contain additionally humectants, emulsifiers, dispersants, stabilizers, solvents or solubilizers or antiseptics. The composition can be sterilized, for example, by filtration using a bacteria impermeable filter, germicidal formulation in it or irradiation. Alternatively, a aseptic solid composition and dissolve in aseptic water or others aseptic means for injection for use.

A dose can be properly determined in consideration of the activity intensity of the active substance, that is, the substance obtained by the screening procedure of the present disclosure, conditions, age or sex of a subject that the administration receives, etc. Preferably, the dose may calculated according to a route as an amount that provides a serum concentration around the tumor or concentration intratumoral 3 to 30 times, for example, 10 times greater than one drug concentration that inhibits 50% of the activity or expression of the polypeptide of the present invention. For example, the oral administration dose in adults (60 kg weight body) is usually about 0.1 to 100 mg / day, preferably 0.1 to 50 mg / day. The dose in the administration parenteral is 0.01 to 50 mg / day, preferably 0.01 to 10 mg / day, in terms of an injection.

A therapeutic target for the composition pharmaceutical is a test subject in which the presence of the polynucleotide of the present disclosure and / or the polypeptide of the present disclosure. The substance that inhibits the polypeptide of the present disclosure kills cells that have transformed due to the fusion polynucleotide of EML4-ALK. Therefore, the substance that inhibits the Polypeptide of the present disclosure serves as a therapeutic agent effective for cancer (particularly, lung cancer) that is shows that it is positive for the polynucleotide of the present invention.

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Examples

The present invention will be described in detail at continued by examples, but the present inventions do not They are limited to these examples. In addition, unless indicated Otherwise, the process of the present invention can be carried carried out according to publicly known procedures. Therefore, the commercially available reagents and kits can be used according to Commercial product instructions.

A full length ALK cDNA was kindly supplied by Dr. Steve Morris of St. Jude Children's Research Hospital. In addition, this research project was approved by the ethical committee for the analysis study of genes at the Jichi University of Medicine.

Anti-ALK antibody phosphorylated and the anti-ALK antibody used were produced by Cell Signaling Tecnology Inc. and NEOMARKERS Inc., respectively.

Example 1 Isolation of the fusion polynucleotide of EML4-ALK v1 (1) Construction of the cDNA library

Using an RNA purification kit (RNeasy Mini Column; Qiagen Inc.), the RNA was extracted from an excised lung adenocarcinoma specimen of a 62-year-old man who gave informed consent and the cDNA was synthesized using reverse transcriptase (Power Script Reverse Transcriptase) and primers (an oligonucleotide of SEQ ID NO: 42 and CDS IIA primer) (all from Clontech Inc.). After selectively amplifying the full length cDNA by polymerase chain reaction (PCR) (17 cycles of 98 ° C for 10 seconds and 68 ° C for 6 minutes) using a primer (5'-PCR first IIA; Clontech Inc.) and a polymerase (PrimeSTAR HS DNA polymerase, Takara Bio Inc.), a BstX1 adapter (Invitrogen Co.) was attached to both ends of the cDNA. The cDNA thus obtained was ligated to a retrovirus plasmid and a retrovirus plasmid library was constructed by introducing this plasmid into E. coli DH10B (Invitrogen Inc.). As a result, the plasmid library containing clones, more than 1,500,000 colony forming units in total, has been successfully constructed.

(2) Focus formation test

2 µg of the plasmid was transfected library described above and 0.5 µg of a plasmid for encapsidation (pGP and pE-echo, which were obtained both of Takara Bio Inc.) with BOSC23 encapsidation cells using a transfection reagent. Two days after transfection, the culture supernatant was recovered as a solution of library of recombinant retroviruses, mixed with polybrene (Sigama Inc.) at a concentration of 4 µg / ml and the mixture is added 3T3 mouse cells to MOI (multiplicity of infection) of concentration 0.1. Two days later, the culture supernatant of 3T3 cells were changed by DMEM-F12 medium (Invitrogen Inc.) supplemented with 5% bovine serum (Invitrogen Inc.) and 2 mM L-glutamine, and the cells are they grew 2 more weeks to get 10 or more types of foci transformed. After isolating each clone of 3T3 cells, the culture of the clones continued separately and the genomic DNA of Each clone was extracted. The viral cDNA integrated into each 3T3 clone was amplified and recovered by performing PCR (30 cycles of 98 ° C for 10 seconds and 68 ° C for 6 minutes) using 10 ng of the DNA genomic as template, primer 5'-PCR primer IIA and DNA polymerase (PrimeStar HS DNA polymerase; Takara Bio Inc.) and se cloned into the vector pT7Blue-2.

One of the cDNAs thus obtained had a length of 3926 base pairs (SEQ ID NO: 1) and had a single frame of open reading (from base 271 to 3447 of SEQ ID NO: 1) that encoded a protein that had 1059 amino acid residues (SEQ ID NO: 2; a novel full length sequence). Approximately half of the amino end of SEQ ID NO: 2 (amino acid residues 1-496 of SEQ ID NO: 2) are matched perfectly with amino acid residues 1-496 of protein 4 similar to protein associated with echinoderm microtubules; EML4 (GenBank registration number NM_019063), and on the other hand, about half of the end carboxyl (amino acid residues 497-1059 of SEQ ID NO: 2) matched perfectly with the amino acid sequence anaplastic lymphoma kinase; ALK (GenBank registration number AB209477). Therefore, in the nucleotide sequence of SEQ ID NO: 1, 99.9% of bases 35-1759 mated with bases 1-1725 of the reported human EML4 cDNA, and bases 1760-3677 of SEQ ID NO: 1 were paired with bases 3613-5530 of human ALK cDNA at 99.9% Although the respective sequences are a bit different from informed base sequences, none of these differences leads to the substitution of amino acids and therefore can be They may be polymorphism of gene sequences. Of the previous results, it was believed that the cDNA present was a cDNA fused between a part of cDNA of EML4 and a part of cDNA of ALK In addition, the cDNA obtained (SEQ ID NO: 1) contained a domain of ALK tyrosine kinase.

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Example 2 Confirmation of the fusion polynucleotide of EML4-ALK v1

The EML4 gene and the ALK gene in humans are both mapped on the short arm of the second chromosome in opposite directions (head to head address). To produce the EML4-ALK fusion polynucleotide found in example 1 it is necessary to cut the respective genomes in the intron downstream of exon 13 of the EML4 gene and intron waters above exon 21 of the ALK gene and religionize them with the gene in the reverse direction. To investigate this directly, the PCR (after 94 ° C for 1 minute, 40 cycles of 98 ° C for 20 seconds and 68 ° C for 9 minutes) was carried out using primers that had the base sequences of SEQ ID NO: 40 (one sequence designed on the transcription chain of the 3 'end of exon 13 of the EML4 gene) and SEQ ID NO: 36 (a sequence designed in the chain complementary to exon 21 of the ALK gene), genomic DNA templates of a patient (ID 33) and the genomic control DNA (the genomic DNA of peripheral monocytes of normal healthy female (46, XX)) and DNA polymerase (LA Taq polymerase; Takara Bio Inc.).

As a result, as shown in Fig. 1, it is obtained a PCR product that had approximately 4 kbp of length only with the genomic DNA of the present patient. That is to say, it was confirmed as expected that at the genomic level the EML4 gene and the ALK gene was cut in the introns and they were religious in the reverse direction. In addition, this PCR product of approximately 4 kbp was cloned into a pT7Blue-2 vector (Novagen Inc.) and the total base sequence was determined. It was obvious that the cuts were located approximately 3.6 kbp downstream of exon 13 of the EML4 gene and 297 bp upstream of exon 21 of the ALK gene. The base sequence is shown in SEQ ID NO: 4. SEQ ID NO: 4 is a genomic sequence that includes the melting point of the EML4 gene and the ALK gene.

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Example 3 EML4-ALK fusion polynucleotide screening in clinical specimens (1) Detection of fusion polynucleotide from EML4-ALK using cDNA

The cDNAs were synthesized from 33 cases of clinical specimens (specimens removed from lung cancer from non-small cells) including the case (ID 33) used herein Example 1 and 2 and peripheral monocytes of a case of a subject normal healthy (46, XX).

To detect the cDNA of the polynucleotide of fusion of EML4-ALK v1, PCR (50 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds and 72 ° C for 1 minute) was carried out using a quantitative PCR kit (QuantiTect SYBR Green; Qiagen Inc.), cDNAs as substrates prepared from clinical specimens and the healthy subject normal described above and oligonucleotides of SEQ ID NO: 8 and 9 as primers. Using the same specimens, the amplifications by PCR of the cDNA of glyceraldehyde-3-phosphate dehydrogenase (hereinafter GAPDH) were tested as a control. To detect the GAPDH cDNA, the oligonucleotides that are constituted by the nucleotide sequences represented by SEQ ID NO: 10 and 11 were used as primers. The samples respectively amplified were electrophoresed with a DNA size marker (marker: 50 bp scale, Invitrogen Inc.). As a result, as shown at the top of the Fig. 2, in the 3 cases in total that include ID 33, the EML4-ALK v1 fusion polynucleotide cDNA. In addition, in all cases analyzed, a clearly confirmed amplification of the GAPDH cDNA (bottom of Fig. 2). In addition, the base sequence of the PCR products was analyzed identified in the cases of ID 20 and ID 39 and the result confirmed that the sequence was identical to that of ID 33 (the 247 bp that include the melting point of the EML4 gene and the ALK gene. SEQ ID NO: 14). That is, the result of the analysis of the 33 cases of non-small cell lung cancer confirmed that the fusion of EML4 gene and ALK gene occurs in 9.1% of cases (3/33 cases).

It has been known that the mutation in the EGFR gene is One of the causes of lung cancer. In the 33 specimens of cases analyzed as described above, the analysis of the presence of an anomaly in the base sequence of the EGFR gene according to the known procedure confirmed a partial deletion of the exon 19 in 6 cases. Cases that have the EGFR gene mutation and positive cases for the fusion polynucleotide of EML4-ALK belonged to different subgroups. Is say, existing therapeutic agents are expected to lung cancer, which are the therapeutic agents for cancer of lung produced by the mutation in the EGFR gene, are not effective for lung cancer patients who are positive for the EML4-ALK fusion polynucleotide.

Therefore, the 33 cases analyzed as described above they underwent the investigation whether the full length ALK gene existed as if it were found that It existed in 8 cases. The specimens of the 7 cases among these 8 cases did not contain the fusion polynucleotide of EML4-ALK (i.e., the full-length ALK gene it did not exist in 2 cases among the 3 cases in which the EML4-ALK fusion polynucleotide).

In addition, analyzes of 42 cases from other cases of non-small cell lung cancer by a procedure similar as described above gave PCR products of approximately 1 kbp in 4.8% of cases (2/42 cases), which are longer than those detected in cases of cases ID 33, ID 20 and ID 39. These were cloned into the vector pT7Blue-2 and the base sequence was analyzed. The results indicated that these were not exon 13 of the EML4 gene, but a fusion product of exon 20 of the EML4 gene and exon 21 of the ALK gene (SEQ ID NO: 3). That is, in these products the fragment of the merged ALK gene was the same, but the cleavage point in the EML4 was different. The cDNA sequence of exon fusion gene 1-20 of the EML4 gene and exon 21-30 of the ALK gene, which contains the melting point of the EML gene and the ALK gene found in SEQ ID NO: 3, is shown in SEQ ID NO: 6, and the sequence of Amino acids of the polypeptide thus encoded are shown in SEQ ID NO: 7. In the present description, the polynucleotide encoding the protein constituted by the polypeptide represented by SEQ ID NO: 7 is called the EML4-ALK fusion polynucleotide v2. The plasmid produced here in which a partial fragment of the EML4-ALK v2 fusion polynucleotide is cloned it designates EML4-ALKv2 partial / pT7Blue-2.

(2) Detection of fusion polynucleotide from EML4-ALK v1 using genomic DNA

It turned out that the presence of the polynucleotide of EML4-ALK fusion can be detected in samples obtained from the test subjects by PCR using the samples of Genomic DNA extracted from clinical specimens (especially the lung tissue) of the test subjects as Example 2. By both, as shown below, the detection of EML4-ALK v1 fusion polynucleotide was tested using various primers. Using 1 ng of the vector pT7Blue-2 as a mold, in which the product was cloned of approximately 4 kbp PCR produced as described above, and using a set of primers [SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:  24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, or SEQ ID NO: 33 and SEQ ID NO: 34], the PCR (30 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute) carried out by means of a DNA polymerase (rTaq DNA polymerase; Takara Bio Inc.). The results indicated that in each PCR amplified a single stranded DNA fragment that had a size expected (about 270 to 380 bp). From the results It is evident that the detection of the polynucleotide of EML4-ALK v1 fusion is possible by carrying out the PCR using genomic DNA extracted from clinical specimens as a mold and various sets of primers that are thought to specifically detect the presence of the fusion polynucleotide of EML4-ALK v1.

(3) Detection of fusion polynucleotide from EML4-ALK v2 using genomic DNA

An oligonucleotide was designed that has the base sequence represented by SEQ ID NO: 35 in exon 20 of EML4 and an oligonucleotide that has the base sequence represented by SEQ ID NO: 36 on the complementary side of the exon ALK 21 as sense primer and antisense primer, respectively. Using these and using a patient's genomic DNA (ID # KL-3121) as a template in which the cDNA was detected of EML4-ALK v2 fusion polynucleotide, PCR (after 94 ° C for 1 minute, 40 cycles of 98 ° C for 20 seconds and 68 ° C for 6 minutes) was carried out with a DNA polymerase (LA Taq polymerase; Takara Bio Inc.). As a result obtained a PCR product of approximately 850 bp. The product of PCR was cloned into the pT7Blue-2 vector, the sequence of bases was determined and a sequence of 853 bp shown was obtained in SEQ ID NO: 5. Sequence analysis revealed that all 35 bp located at the 3 'end of exon 20 of EML4 and the 544 bp of the intron sequence downstream of exon 20 of EML4 were ligated at 233 bp of the intron sequence upstream of exon 21 of ALK and the 41 bp located at the 5 'end of exon 21 of ALK. That is, it is evident that genome excision occurred in a location 544 bp downstream of exon 20 of EML4 and in a location 233 bp upstream of exon 21 of ALK. Using 1 ng of pT7Blue-2 vector as a template, in which the clone was cloned 853 bp PCR product prepared as described above, and using a set of primers [SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 18, SEQ ID NO: 41 and SEQ ID NO: 20, SEQ ID NO: 43 and SEQ ID NO: 22, SEQ ID NO: 45 and SEQ ID NO: 24, SEQ ID NO: 47 and SEQ ID NO: 26, SEQ ID NO: 49 and SEQ ID NO: 28, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, or SEQ ID NO: 55 and SEQ ID NO: 34], the PCR reaction was carried out under them conditions than those of Example 3 (2). As a result, in each PCR was amplified a single stranded DNA fragment that had the expected size (approximately 270 to 380 bp). Of the previous results it turns out that the detection of the presence of EML4-ALK v2 fusion polynucleotide is possible carrying out the PCR using the genomic DNA extracted from the clinical specimens such as mold and primer sets that are think that they specifically detect the fusion polynucleotide of EML4-ALK v2.

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Example 4 Procedure to detect mRNA of the fusion polynucleotide of EML4-ALK (1) Construction of the polypeptide expression vector of fusion of EML4-ALK v1 and cloning of EML4-ALK v2 fusion polynucleotide

From the clone in which the clone was cloned EML4-ALK v1 fusion polynucleotide in the positive direction (designated EML4-ALK v1 / pT7Blue-2), the insert was removed by digesting with restriction enzymes EcoRI and SalI and was subcloned at sites EcoRI-SalI of pMXS (JBC 275, 24945-24952, 2000). This one was designated EML4-ALK v1 / pMS. Therefore, the cDNA of the bases 277-3450 of SEQ ID NO: 1 which has the sites in both ends that are recognized by the restriction enzyme EcoRI was amplified by carrying out the PCR (25 cycles of 98 ° C during  10 seconds, 68 ° C for 5 minutes) using the plasmid EML4-ALK v1 / pT7Blue-2 as a mold and oligonucleotides that are constituted by the base sequence represented by SEQ ID NO: 59 and SEQ ID NO: 60 as primers and a DNA polymerase (PrimeStar HS DNA polymerase). After digesting with EcoRI, this was introduced into the EcoRI site of a vector of pcDNA3 expression, which is modified so that the insert can express with a FLAG mark added to the N end to produce a expression plasmid (FLAG-EML4-ALKv1 / pcDNA3) for the EML4-ALK v1 fusion polypeptide that has the FLAG mark at end N (hereinafter referred to as FLAG-EML4-ALKv 1). In addition, the cDNA of FLAG-EML4-ALKv 1 was removed from this vector digesting with restriction enzymes HindIII and XbaI, and after converting both ends into blunt, an adapter EcoRI-NotI-BamHI (Takara Bio Inc.) It linked to both ends. The product was introduced on the site EcoRI of an expression vector, whereby the inserted cDNA and the CD8 cell surface antigen can be expressed thereto time (bicistronic vector pMX-iresCD8; J. Biol. Chem. 2001, vol. 276, p39012-39020) to produce a expression vector that expresses both FLAG-EML4-ALKv1 as CD8. This is appointed FLAG-EML4-ALKv1 / pMX-iresCD8.

The fusion polynucleotide of EML4-ALK v2 was cloned as follows.

Using an EML4 full length polynucleotide cloned into pT7Blue-2 according to "Genomics", (USA), 2000, vol. 68, p. 348-350, as a template to obtain a polynucleotide fragment encoding EML4 of the EML4-ALK v2 fusion polynucleotide, and using an oligonucleotide represented by SEQ ID NO: 57, in which an EcoRI cleavage sequence is attached to the end 5 'of the ATG initiation codon of exon 1 of the EML4 gene, and an oligonucleotide represented by SEQ ID NO: 58, whose sequence is constituted by 10 bases of the 5' end of the antisense sequence of exon 21 of the ALK gene fused to end 5 'of the antisense sequence at the 20 bases of the 3' end of exon 20 of the EML4 gene, respectively, as sense and antisense primers, and using a DNA polymerase ( Pyrobest DNA polymerase; Takara Bio Inc.), PCR (25 cycles of 94 ° C for 20 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute) was carried out to obtain a PCR product of approximately 2260 bp. This product was designated PCR product A.

While using the EML-ALKv1 / pTBlue-2 produced in the present Example 4 (1) as a template to obtain the fragment of ALK polynucleotide of the fusion polynucleotide of EML4-ALK v2, and using an oligonucleotide represented by SEQ ID NO: 101, the sequence that is constituted by the 10 bases of the sequence felt at the 3 'end of the exon 20 of the EML4 gene fused to the 5 'end of the 20 bases of the sequence felt at the 5 'end of exon 21 of the ALK gene and a oligonucleotide represented by SEQ ID NO: 102, in which the Xba I cleavage sequence is attached to the 5 'end of the area of the antisense sequence containing the termination codon present in exon 30 of the ALK gene as sense and antisense primers, respectively, the PCR was carried out under the same condition as to obtain the PCR product A to obtain a PCR product of approximately 1700 bp. This was designated the PCR product B.

The PCR products A and B described above were mixed and hybridization and extension reactions were carried out (3 cycles of 94 ° C for 1 minute, 55 ° C for 30 seconds and 72 ° C for 2 minutes and 30 seconds) using a DNA polymerase (DNA polymerase Pyrobest ; Takara Bio Inc.) to obtain a product of approximately 4000 bp. This product was cloned by TA into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen Inc.) and the base sequence was analyzed. It was obtained, as shown in SEQ ID NO: 6, which consisted of 2242 bases from the ATG initiation codon of the EML4 gene to exon 20 and 1691 bases from exon 21 of the ALK gene to the termination codon of exon 30 .

(2) Procedure to detect polynucleotide mRNA from fusion of EML4-ALK v1 and v2

Using 1 ng of FLAG-EML4-ALKv1 / pMX-iresCD8 described in (1) as a fusion polynucleotide template of EML4-ALK v1 and 1 ng of EML4-ALKv2 partial / pT7Blue-2 in example 3 as a template for EML4-ALK v2 fusion polynucleotide, and using a set of primers [set of primers for the EML4-ALK v1 fusion polynucleotide; (SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, or SEQ ID NO: 79 and SEQ ID NO: 80) and the primer set for the EML4-ALK v2 fusion polynucleotide; (SEQ ID NO: 81 and SEQ ID NO: 62, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 68, SEQ ID NO: 87 and SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 70, SEQ ID NO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 74, SEQ ID NO: 95 and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, or SEQ ID NO: 99 and SEQ ID NO: 100)], the PCR (30 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute) was carried out by a DNA polymerase (DNA polymerase rTaq; Takara Bio Inc.). As a result, in the whole set of primers a single stranded DNA fragment was amplified, having each the expected fragment (approximately 260 to 350 bp). From The previous results confirmed that the detection of presence of the EML4-ALK fusion polynucleotide v1 and / or v2 is possible by carrying out RT-PCR according to the present example using mRNA extracted from subject samples Test as a mold.

The results of Example 3 and 4 (2) described above indicated that the presence of EML4-ALK v1 and v2 fusion polynucleotide can be detected using both cDNA and genomic DNA prepared from the clinical specimens obtained from test subjects. This fact suggests that patients who have the fusion polynucleotide of EML4-ALK can be selected and it can practice a tailor-made treatment so patients to be treated by administering inhibitors of EML4-ALK fusion polynucleotide and / or Polypeptide are selected beforehand and then treated.

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Example 5 Fusion polynucleotide detection of EML4-ALK v1 in sputum (1) Production of mouse BA / F3 cells expressing FLAG-EML4-ALKv1

A recombinant retrovirus was produced by a procedure similar to the one described above using FLAG-EML4-ALKv1 / pMX-iresCD8 and a white vector (pMX-iresCD8) and with it infected BA / F3 cells of the mouse lymphatic cell line. Cells expressing CD8 on the cell surface were purified  simply using magnetic beads reagent for separation of cells and a purification column (magnetic beads attached to anti-CD8 monoclonal antibody and column of MiniMACS purification, both from Miltenyi Biotec Inc.).

(2) Detection of fusion polynucleotide from EML4-ALK v1 in sputum

After mixing sputum samples from subjects normal healthy with BA / F3 cells expressing polynucleotide of EML4-ALK v1 merger (hereinafter referred to as BA / F3 cells expressing v1) at 0 / ml, 10 cells / ml, 100 cells / ml, 1000 cells / ml and 10,000 cells / ml, the cDNA is synthesized by the standard procedure. The presence of EML4-ALK v1 fusion polynucleotide in sputum is examined by carrying out a PCR reaction (50 ° C for 2 minutes, 95 minutes for 15 minutes and additionally 40 cycles of the next reaction (94 ° C for 15 seconds, 60 ° C for 30 seconds and 72 ° C for 1 minute)) using the cDNA described formerly as a substrate, and the oligonucleotides that are constituted by the sequence of bases represented by SEQ ID NO: 8 and SEQ ID NO: 9 as primers and a quantitative PCR kit (QuantiTect SYBR Green; Qiagen Inc.), and products were obtained from PCR As a result, in each case, except at 0 / ml, it could be confirmed the presence of the fusion polynucleotide of EML4-ALK v1.

Conventionally, the cytopathological examination using sputum samples has been an important procedure of diagnosis for the diagnosis of lung cancer. This diagnosis for lung cancer is based on the presence of atypical cells in sputum, but a diagnosis cannot be made reliable unless there are many cells in the sputum lung cancer However, most of the time, such cases were already in the advanced state and it was almost impossible practice an effective early diagnosis for lung cancer. According the present invention, if the fusion polynucleotide of EML4-ALK is present in sputum, it is clear that It can be detected by PCR even if there is a minimum quantity.

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Example 6 Transformability and tumorigenicity investigation of EML4-ALK v1 fusion polypeptide (1) Analysis of the fusion polypeptide of EML4-ALK v1

The EML4-ALK (K589M) / pMXS, in the that amino acid 589 (ATP binding site), a lysine residue, of the EML4-ALK v1 fusion polypeptide is replaced with methionine, was produced using EML4-ALKv1 / pMXS as a substrate and using a kit introduction of mutations (site-directed mutagenesis kit QuickChange; Stratagene Inc.). In the reaction they were used oligonucleotides of SEQ ID NO: 103 and SEQ ID NO: 104. The ALK cDNA (Morris, SW et al., Science. March 4, 1994; 263 (5151): 1281-4) was cloned with a retrovirus vector pMXS according to the standard procedure (designated ALK / pMXS).

The EML4-ALKv1 / pMXS described above, ALK / pMXS, a plasmid expressing EML4-ALK
(K589M) / pMXS and a white vector without inserted cDNA (pMXS) were transfected into 3T3 fibroblast cells by the calcium phosphate procedure and cultured for 21 days. As shown in the upper part of Fig. 3, only many foci of transformation were observed when the EML4-ALK v1 fusion polypeptide expressing the vector was transfected. The scale bar indicates 100 µm. In addition, the same transfected 3T3 cells were inoculated subcutaneously to nude mice at 5 x 10 5 cells / mouse and observed for 20 days. It also turned out that the tumor only formed when the EML4-ALK v1 fusion polypeptide expressing cells was inoculated. The tumor formation numbers (the number of 3T3 cell inoculation sites and the number of tumor formation between them) are as follows. The tumor formation number of the full-length expression of ALK was 0 in 8, while the number of tumor formation in the EML.4-ALK v1 fusion polypeptide expressing cells was 8 in 8. In addition, the number of tumor formation of EML4-ALK (K589M) expressing cells was 0 in 8. These results demonstrate that since expression of the full-length ALK polypeptide does not induce tumor, but the EML4-ALK v1 fusion polypeptide is tumorigenic. , the EML4-ALK v1 fusion polynucleotide is the causative gene of cancer. Therefore, because the tumorigenicity of EML4-ALK was not observed in EML4-ALK (K589M), it would appear that the tumorigenicity depended on the kinase activity. Hereinafter, 3T3 cells in which the EML4-ALK v1 fusion polypeptide is expressed are designated cells
3Q3 expressing v1.

(2) Analysis of various deletion mutants of the EML4-ALK v1 fusion polynucleotide

The various deletion mutants (mutant of ΔBasic deletion (amino acids were deleted 31-140 of the fusion polypeptide of EML4-ALK v1), ΔHELP deletion mutant (se deleted amino acids 220-296 from EML4-ALK v1) fusion polypeptide, mutant of ΔWD deletion (amino acids were deleted 305-475 of the fusion polypeptide of EML4-ALK v1)) of the EML4 part of the polynucleotide EML4-ALK v1 fusion were prepared by PCR reaction using FLAG-EML4-ALKv 1 / pMX-iresCD8 as a mold and using a kit cloning (site-directed mutagenesis based on ExSite PCR; Stratagene Inc.). Using these deletion mutant plasmids, prepared retrovirus solutions using a procedure similar to that of Example 1 to obtain infected 3T3 cells. These respective infected cells were inoculated subcutaneously to nude mice to investigate tumorigenicity and found that the tumors were formed by 3T3 cells that express the mutant deletion ΔHELP and the deletion mutant ΔWD. He number forming tumor of respective 3T3 cells expressing the deletion mutant ΔBasic, the deletion mutant ΔHELP and the deletion mutant ΔWD was 0 between 8, 7 between 8 and 8 between 8, respectively. How no training was observed of tumors in the ΔBasic deletion mutant, it is shown that amino acids 31-140 of the fusion polypeptide of EML4-ALK v1 are important in tumorigenesis. How the fusion polypeptide of EML4-ALK v2, like the fusion polypeptide of EML4-ALK v1, contains amino acids 31-140 mentioned above and the region of ALK kinase, the fusion polynucleotide of EML4-ALK v2, like the polynucleotide of EML4-ALK fusion, is the causal cancer gene that encodes the polypeptide that has the transformability and tumorigenicity for 3T3 cells.

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Example 7 Screening procedure for polypeptide inhibitors EML4-ALK fusion (1) Preparation of the fusion polypeptide of EML4-ALK v1

BA / F3 cells expressing v1 (Example 5 (1)) were grown in RPM1640 medium containing 10% of fetal bovine serum to obtain 2.7 x 10 9 cells. After wash 3 times with PBS, the cells were lysed in a solution of lysis (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, Triton 1% X100, 5 mM EDTA, 5 mM EGTA, 1 mM NaVO 4, 1 mM DTT and complete mixture of protease inhibitor). The fusion polypeptide of EML4-ALK v1 present in the supernatant obtained after centrifugation it was purified using affinity gel ANTI-FLAG M2 (SIGMA-ALDRICH Inc.) according to the procedure described in the information document of the product. For washing and elution the solution of Wash (50 mM Tris-HCl (pH 7.4), 250 mM NaCl, Brij35 0.05%, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO 4, 1 mM DTT, complete) and elution solution (Tris-HCl 50 mM (pH 7.4), 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 0.5 mg / ml of FLAG peptide), respectively. The immunoblot using anti-ALK antibody and antibody anti-FLAG M2 (SIGMA-ALDRICH Inc.) and silver staining were carried out for the eluate to detect the EML4-ALK v1 fusion polypeptide. Be demonstrated that the EML4-ALK fusion polypeptide v1 can be prepared by this procedure.

(2) Detection of in vitro kinase activity of the EML4-ALK v1 fusion polypeptide

The fusion polypeptide of EML4-ALK v1 purified as described previously diluted in a reaction solution (15 mM Tris-HCl (pH 7.4), 0.25 mM MgCl 2, 0.01% Tween-20, 2 mM DTT) and then not added ATP or 20 µM ATP was added. The respective mixtures were made react at room temperature for 1 hour. After the reaction, the EML4-ALK v1 fusion polypeptide autophosphorylated and fusion polypeptide of EML4-ALK v1 were detected by immunoblot using phosphorylated anti-ALK antibody that recognizes specifically the phosphorylated product in the tyrosine residue 1604 of ALK, and anti-ALK antibody, and quantified by an image analysis system (VersaDoc Imaging System; Bio-Rad Inc.). The amount of phosphorylation was calibrated by dividing the polypeptide count of fusion of self-phosphory EML4-ALK v1 between the EML4-ALK v1 fusion polypeptide count. As a result, the fusion polypeptide band of EML4-ALK v1 autophosphorylated was detected in the location of approximately 130 kDa under the condition of the addition of ATP, and the amount of phosphorylation increased approximately 205 times compared to the non-addition of ATP.

In addition, phosphorylation activity against a peptide substrate was investigated using a kinase activity detection kit (HTRF KinEASE-TK; Cisbio Inc.). Using TK subtract 1, which was included in the kit, as a substrate and after not adding ATP or adding 100 µM ATP, the mixtures were reacted at room temperature for 1 hour and the HTRF count was detected as recommended by the kit manufacturer As a result, it is clear that the HTRF count (ie, phosphorylation of the peptide substrate) increased approximately 12 times by the addition of ATP compared to the non-addition of ATP. As shown above, the in vitro kinase activity of the EML4-ALK v1 fusion polypeptide can be detected using phosphorylated anti-ALK antibody and the kinase activity detection kit.

(3) Inhibitory effect of compounds against in vitro kinase activity of EML4-ALK v1 fusion polypeptide

The inhibitory effect of compound AD against in vitro kinase activity of the EML4-ALK v1 fusion polypeptide was investigated using phosphorylated anti-ALK antibody and the kinase activity detection kit. The respective compounds were added to the reaction solution containing the EML4-ALK v1 fusion polypeptide at a final concentration of 10 µM or 10 nM, and then the reaction was carried out with or without the addition of ATP. The rest of the operations were carried out according to the procedure (2) described above. In the absence of the compound it was assumed that the phosphorylation count without the addition of ATP and with the addition of ATP was 100% inhibition and 0% inhibition, respectively. Inhibition (%) of the kinase activity of the EML4-ALK v1 fusion polypeptide by a compound was calculated by the following formula.

[Inhibition of kinase activity (%) by a compound = (1- [phosphorylation count when
the compound and ATP-phosphorylation count were added when the compound was not added and ATP] / [phosphorylation count was not added when the compound was not added and ATP-phosphorylation count was added when the compound was not added and not added ATP]) x 100

As a result it was found that the compound A-D inhibited phosphorylation activity of purified EML4-ALK v1 fusion polypeptide itself itself and the peptide substrate (Table 1). Compound A and B could be selected as substances that inhibited the activity of EML4-ALK v1 50% or more fusion polypeptide at a concentration of 10 µM or less, and compound C and D could be selected as substances that inhibited the activity of EML4-ALK v1 50% or more fusion polypeptide at a concentration of 0.1 µM or less.

TABLE 1

one

The above results indicated that screening (an in vitro type screening) of a substance that inhibits the activity of the polypeptide of the present invention could be performed by preparing the EML4-ALK fusion polypeptide and using the in vitro kinase activity as an index.

(4) Inhibitory effect of polypeptide inhibitors of EML4-ALK v1 fusion on autophosphorylation intracellular (4-1) BA / F3 cells

Compound A (1 µM, 5 µM and 10 µM) BA / F3 cells expressing v1 was added to the culture medium (Example 5 (1)) or was not added, and cultured for 3 hours. In addition, BA / F3 cells that express FLAG-EML4-ALKv1 (K589M) se produced using the vector pMX-iresCD8 in which integrated EML4-ALK (K589M) so that it could FLAG was added, and they were grown. After cultivation, the cells are counted and the tyrosine phosphorylation level of the polypeptide of EML4-ALK v1 fusion was measured by immunoblot using anti-ALK antibody phosphorylated In addition, for the same transfer membrane, carried out antibody immunoblot analysis FLAG anti-brand (Eastman Kodak Inc.) and the amount of total fusion polypeptide protein from EML4-ALK v1 attached to FLAG. As shown in the upper part of Fig. 4, the phosphorylation level of the EML4-ALK v1 fusion polypeptide tyrosine se detected in BA / F3 cells expressing v1, but the tyrosine phosphorylation when EML4-ALK was expressed  (K589M). This fact indicates that the tyrosine phosphorylation of EML4-ALK v1 fusion polypeptide detected in BA / F3 cells is an autophosphorylation by the polypeptide itself EML4-ALK v1 fusion. Therefore, it has been confirmed that compound A inhibits intracellular autophosphorylation of the EML4-ALK v1 fusion polypeptide in one mode concentration dependent. In addition, it has been shown that the amount of expression of the polypeptide protein itself EML4-ALK v1 merger has been almost constant in all samples (Fig. 4, bottom).

The inhibitory effect of the compound B-D on intracellular autophosphorylation is examined in a similar manner as described above. Without However, the cultivation time after the addition of the compounds was 6 hours, and the total protein amount of the EML4-ALK v1 fusion polypeptide was measured using anti-ALK antibody. In addition, the amount of phosphorylation was calculated by quantifying as in the Example 7 (2). Therefore, for compound A, the amount of phosphorylation was calculated by quantifying in the experiment under this condition. The inhibition rate was calculated from the amount of phosphorylation when the compound was added, using as a control the value when the compound was not added (the compound solvent, DMSO) (0% inhibition). Each compound clearly inhibited the kinase activity of the fusion polypeptide of EML4-ALK v1 in BA / F3 cells (Table 2). He compound A and B can be selected as the substance that inhibits EML4-ALK fusion polypeptide activity v1 50% or more at a concentration of 10 µM or less, and the compound C and D can be selected as the substance that inhibits the EML4-ALK v1 fusion polypeptide activity 50% or more at a concentration of 0.1 µM or less, and it has been shown that substances that inhibit the activity of Polypeptide of the present invention can be screened (screened based in cells).

(4-2) 3T3 cells

In a similar way to in (4-1), except that the A-D compounds were added to 3T3 cells expressing v1 (Example 6 (1)) at 10 µM or 10 nM and grown for 4 hours, the amount of tyrosine phosphorylation of the fusion polypeptide of EML4-ALK v1 and the total amount of protein in the EML4-ALK v1 fusion polypeptide and was calculated the rate of inhibition of the respective compounds in the intracellular kinase activity. Each compound clearly inhibited the kinase activity of the fusion polypeptide of EML4-ALK v1 in 3T3 cells expressing v1 (Table 2). It is evident that various cells such as  BA / F3 cells, 3T3 cells and the like as cells expressing the polypeptide of the present invention in the screening process based on cells of the present invention.

TABLE 2

2

The previous results indicated that the compound that inhibits the kinase activity of the polypeptide of EML4-ALK v1 fusion can be obtained using as autophosphorylation index in cells expressing the EML4-ALK v1 fusion polypeptide.

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Example 8 Growth inhibitory effect of inhibitors of EML4-ALK fusion polypeptide on cells that express EML4-ALK fusion polynucleotide v1 (1) Growth potential of BA / F3 expressing v1

For the growth of BA / F3 cells that express only the CD8 protein (Example 5 (1)), the BA / F3 cells that express CD8, in addition to ALK, the EML4-ALK v1 fusion polypeptide (Example 5 (1) ) or the EML4-ALK (K589M) deficient in kinase activity, the change in the number of cells was counted starting at 8 x 10 5 cells over time in the presence or absence of an IL growth factor -3. The results are shown in Fig. 5. BA / F3 cells expressing v1 can grow with or without IL-3. However, BA / F3 cells that only express CD8 grew in the presence of IL-3, but died quickly when IL-3 was removed. This fact indicates that the EML4-ALK v1 fusion polynucleotide has an activity as an oncogene. In addition, cells expressing full-length human ALK and BA / F3 cells expressing EML4-ALK (K589M) lacking kinase activity died similarly in the absence of IL-3. These results indicate that the cells obtain the growth potential, even in the absence of the growth factor, by expressing the EML4-ALK v1 fusion polypeptide and that the growth potential depends on the kinase activity of the EML4-ALK fusion polypeptide. v1. BA / F3 cells expressing full-length ALK were obtained according to
Example 5 (1).

(2) Inhibitor growth inhibitory effect of EML4-ALK fusion polypeptide on cells BA / F3 expressing v1

Next, compound A, which is a substance that inhibits the fusion polypeptide of EML4-ALK v1, was added to BA / F3 cells that obtained the potential for independent growth of IL-3 expressing the fusion polypeptide of EML4-ALK v1, and its effect on the cell growth When compound A was added at 1 µM, 5 µM or 10 µM, or not (0 µM) was added to the control, measured BA / F3 cells expressing CD8, which grow in presence of IL-3, and cell growth, the cells were able to grow although a slight inhibition of growth as shown in Fig. 6 (a). On the other hand, when compound A was added to BA / F3 cells expressing v1 that were growing in the absence of IL-3, the cell growth was markedly inhibited by dependence on concentration of compound A and cell death was induced as shown in Fig. 6 (b). That is, it was confirmed that the cells, which grow independently in the polynucleotide of fusion of EML4-ALK (oncogene), were destroyed by an EML4-ALK fusion polypeptide inhibitor v1.

(3) Inhibitory effect of polypeptide inhibitors of EML4-ALK fusion on growth independent of the anchoring of cells expressing the polypeptide of fusion of EML4-ALK v1 and the ALK polypeptide of full length

It is known that measuring cell growth Anchor independent (colony procedure, etc.) is a system to investigate an antitumor effect (pharmacological effect) of compounds (Clinical Oncology, second edition, Cancer and Chemotherapy Publishers Inc.). Instead of the procedure of colonies, there is a following procedure that uses spheroid plaques to measure the growth of unbound cells.

3T3 cells that express v1 (Example 6 (1)) and one of the human glioma cells that express the full-length ALK polypeptide and that do not express endogenously EML4-ALK fusion polynucleotide, U-87MG cells, are planted in a 96-well spheroid plate (Sumilon Celltight Spheroid 96U, Sumitomo Bakelite Inc.) at 3000 cells per well in a medium containing 10% fetal bovine serum (DMEM for 3T3 cells expressing v1 and RPMI 1640 for U-87MG ). Under 5% CO2, the cells were grown overnight at 37 ° C and then compound A or B was added at a final concentration of 10 µM, compound C or D was added at a final concentration of 10 nM and as a negative control the solvent of the compounds, DMSO, was added to give the same concentration as the compounds. At the same time, cells were counted before drug addition (day 0). Then, the cells were grown under 5% CO2, at 37 ° C for 3 days, mixed with a reagent to measure the number of cells (CellTiter-Glo ™ luminescent viability test; Promega Inc.), they were stirred for 20 minutes and the measurements (day 3) were carried out using a luminometer (ML3000 microtiter plate luminometer; Dynatech Laboratories Inc.). The results show that all compounds had growth inhibitory activity in 3T3 cells expressing v1, but had almost no inhibitory activity in U-87MG cells. The rate of inhibition of the compounds was calculated assuming that the number of cells on day 0 and day 3 was 100% of the inhibition and 0% of the inhibition, respectively
(Table 3).

TABLE 3

3

The previous results indicate that the compound A-D inhibited cell growth independent of the anchoring of 3T3 cells expressing v1 by inhibiting the kinase activity of the fusion polypeptide of EML4-ALK v1. In addition, it is clear that these compounds could not inhibit independent cell growth of the anchor of U-87MG cells expressing the full length ALK polypeptide. These results indicate that fusion polypeptide inhibitors of EML4-ALK can inhibit cell growth cancerous and tumors expressing the fusion polypeptide of EML4-ALK.

(4) Preparation of siRNA for the fusion polynucleotide from EML4-ALK- v1

The siRNAs, which were composed of a transcription chain that consists of the sequences of bases represented by SEQ ID NO: 111, 113, 115, 117, 119 or 121 and a complementary chain that consists of the sequences of bases represented by SEQ ID NO: 112, 114, 116, 118, 120 or 122, were prepared as siRNAs (siRNA-1 to SiRNA-6) that had 100% homology to the area EML4-ALK v1 fusion polypeptide fusion and are expected to have inhibitory activity on the expression of EML4-ALK v1 fusion polypeptide. In addition, the SiRNA composed of a transcription chain that is consisting of the base sequences represented by SEQ ID NO: 123 or 125 and a complementary chain consisting of the base sequence represented by SEQ ID NO: 124 or 126 is designed and prepared as siRNAs (siRNA-7, SiRNA-8) showing 100% homology with the ALK area of EML4-ALK v1 fusion polypeptide and are expected to have an inhibitory effect on the expression of ALK gene, using an siRNA sequence design system (Commercial siDirect (registered trademark) RNAi Inc.). It has been confirmed by the siRNA sequence design system (Commercial siDirect (registered trademark) RNAi Inc.) that base sequences corresponding to siRNAs 1 to siRN-8 does not show 100% homology with the gene other than the EML4-ALK fusion polynucleotide and ALK genes. For control experiments to investigate the influence of non-specific siRNA, the siRNA composed of a chain  of transcription that is constituted by a sequence of bases represented by SEQ ID NO: 127 and a complementary chain that It consists of a sequence of bases represented by SEQ ID NO: 128 was prepared as siRNA (siRNA-9) corresponding to a base sequence not present in cells of mammal. The siRNA-1 is a product of hybridizing SEQ ID NO: 111 (transcription string) and SEQ ID NO: 112 (string complementary), and the siRNA-2 and others are the themselves (Fig. 7). (5) Inhibitory effect of siRNA on expression of mRNA of the EML4-ALK fusion polynucleotide and the ALK gene in cells expressing the fusion polynucleotide of EML4-ALK v1 and ALK full length.

3T3 cells expressing v1 and cells U-87MG were seeded in 12-well plates (IWAKI;  Asahi techno glass corp.) To 50,000 cells and 150,000 cells, respectively. Four hours later, using a reagent of transfection (Lipofectamine RNAiMax; Invitrogen Inc.), the SiRNA-1 to siRNA-9 were prepared to a final concentration of 20 nM and were transfected into cells according to The attached instruction. In addition, as a control samples were prepared of transfection without siRNA. After 72 hours, the total RNA is extracted and cDNA was prepared.

The amount of mRNA expressed from the polynucleotide of EML4-ALK v1 fusion in 3T3 cells expressing  v1 and the amount of mRNA of the ALK gene in cells U-87MG were quantified using a PCR reagent Quantitative (Power SYBR Green PCR Master Mix; Applied Biosystems Inc.) The PCR reaction was carried out as follows: after 10 minutes of incubation at 95 ° C, 45 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds, and then a cycle of 95 ° C for 15 seconds, 60 ° C for 15 seconds and 95 ° C for 15 seconds to complete the reaction. In addition, to calibrate the amount of expression, the amount of gene expression of Mouse cyclophilin B and the human GAPDH gene was quantified similarly for 3T3 cells expressing v1 and cells U-87MG, respectively. The analyzes led to out using a sequence detector (detection system ABI PRISM 7900 sequences; Perkin-Elmer Applied Biosystems Inc.).

The oligonucleotides that are constituted by the base sequences represented by SEQ ID NO: 44 and 48, SEQ ID NO: 50 and 56, SEQ ID NO: 44 and 48 and SEQ ID NO: 12 and 13 were used as sets of primers that specifically recognize the EML4-ALK v1 fusion polynucleotide, the gene of Mouse cyclophilin B, the human ALK gene and the human GAPDH gene. Therefore, to obtain a standard curve to calculate the quantity of respective mRNA, PCR was performed using human genomic DNA (Clontech) as a template for the fusion polynucleotide of EML4-ALK v1, the human ALK gene and the GAPDH gene human, and using mouse genomic DNA (Clontech) as a template for mouse cyclophilin B and primer sets above mentioned under the same condition. How a set of primers corresponding to exon 29 of the ALK polynucleotide human to detect the fusion polynucleotide of EML4-ALK v1, the standard curve can be obtained using Human genomic DNA The amount of polynucleotide expression of fusion of EML4-ALK v1 and the human ALK gene in respective samples were calibrated with the amount of expression of the mouse cyclophilin B gene and the human GAPDH gene to obtain the Amount of expression calibrated. Also, assuming the amount of respective calibrated expression of the fusion polynucleotide of EML4-ALK v1 and the human ALK gene in the absence of siRNA out of 100%, the amount of relative expression of the amount of calibrated expression of the fusion polynucleotide of EML4-ALK v1 and the human ALK gene and the rate of inhibition for expression when transfected the respective siRNAs (Table 4).

As a result, siRNAs 1 to SiRNA-8, which correspond to the polynucleotide EML4-ALK v1 fusion inhibited expression of EML4-ALK 50% fusion polynucleotide or plus. For the human ALK gene, the siRNA-7 and SiRNA-8, which correspond to the human ALK gene, inhibited 50% or more. The negative control, SiRNA-9, did not demonstrate a strong inhibitory effect of expression on the fusion polynucleotide of EML4-ALK and the human ALK gene. This results demonstrate that substances that inhibit fusion polynucleotide expression of EML4-ALK.

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TABLE 4

4

(6) Inhibitory effect of siRNA on expression and autophosphorylation of the fusion polypeptide of EML4-ALK v1 in 3T3 cells expressing v1

By the similar procedure described before, 3T3 cells expressing v1 were plated on 12 plates wells at 50,000 cells per well and 4 hours later transfected siRNA-1 to siRNA-9. In addition, cells in which no siRNA was transfected were prepared. as control After 72 hours of transfection, the medium is removed and autophosphorylation of the fusion polypeptide from EML4-ALK v1 intracellular and the amount of protein of the EML4-ALK v1 fusion polypeptide is quantified by the procedure similar to that of Example 7 (4). Therefore, to confirm that the amount of protein total in each sample was the same for measurement, the protein of Actin was quantified using anti-actin antibody (SIGMA-ALDRICH Inc.). The siRNAs 1 to SiRNA-8 clearly inhibited the expression of EML4-ALK v1 fusion polypeptide and activity of kinase in 3T3 cells expressing v1.

(7) Inhibitory effect of siRNA growth on 3T3 cells expressing v1 and cells expressing ALK in length complete

The speed of growth inhibition cell was calculated with or without transfecting siRNA using the procedure similar to that of Example 8 (3), except that each SiRNA was added beforehand to 96-well spheroidal plates and then 3T3 cells expressing v1 and cells were seeded U-87MG and grown for 3 days.

The results of these experiments are shown. in Table 5. Each siRNA (siRNA-1 to SiRNA-8), which had been shown to have an effect inhibitory on kinase expression and activity of EML4-ALK v1 fusion polypeptide in the Example 8 (5) (6), strongly inhibited independent growth of the anchor of 3T3 cells expressing v1. Because the number of 3T3 cell cells expressing v1, in which transfected siRNA-1, 3, 4 and 5, it was lower in the moment of measurement (day 3) than when the siRNA was transfected (day 0) resulting in the rate of inhibition of growth was greater than 100%, it is believed to have been induced cell death On the other hand, it was shown that SiRNA-7 and siRNA-8 inhibited ALK gene expression in Example 8 (5), but did not inhibit the growth of U-87MG cells that express ALK full length SiRNA-1 a SiRNA-6 showed no growth inhibition in U-87MG cells and the negative control, SiRNA-9 did not inhibit the growth of 3T3 cells which express v1 and U-87MG cells.

TABLE 5

5

From the previous results it is clear that, transfecting siRNA which is against the fusion polynucleotide of EML4-ALK v1 to cancer cells that express the EML4-ALK v1 fusion polynucleotide, the mRNA expression of the fusion polynucleotide of EML4-ALK v1 is inhibited resulting in EML4-ALK fusion polypeptide reduction and inhibition of autophosphorylation causing inhibition of cancer cell growth Therefore, for cells cancers that express full-length ALK, inhibition of growth did not occur when the expression of ALK From the previous results it is clear that the siRNA against EML4-ALK v1 fusion polynucleotide, by example, siRNA-1 to siRNA-8, is useful as a therapeutic agent against the tumor that expresses the EML4-ALK fusion polynucleotide for positive patients for fusion polynucleotide EML4-ALK.

(8) Antitumor test for polypeptide inhibitors of EML4-ALK fusion against 3T3 cells that express v1

3 times were inoculated subcutaneously 10 6 3T3 cell cells expressing v1 suspended in PBS by injection into the back of nude BALB / c male mice of 5 weeks old (Japan Charles River Inc.). 7 days after inoculation the administration of compound C, a EML4-ALK fusion polypeptide inhibitor (Table 1 to 3). The test was performed in the solvent group and the group of compound C, 4 animals per group. Compound C is dissolved in a solvent composed of 10% of 1-methyl-2-pyrrolidinone  (SIGMA-ALDRICH Inc.) / 90% polyethylene glycol 300 (Fluka Inc.) and was administered orally at the dose of 10 mg / kg. The administrations were performed once a day for 14 days and body weight and tumor size were measured every two days. He Tumor volume was calculated using the following formula.

[Tumor volume (mm3)] = [axis major tumor (mm)] x [minor axis of the tumor (mm)] 2 x 0.5

The rate of inhibition of compound C is calculated assuming that the tumor volume of the solvent group on the day of beginning and the day of ending the administration was of 100% inhibition and 0% inhibition, respectively. The results indicated that compound C inhibited the growth of 3T3 cells expressing v1 (tumor) 103%.

The antitumor effect of compound D is investigated for the similar procedure with the following exceptions Administration of compound D began 6 days later of inoculation and was carried out once a day for 10 days, and then the size of the tumor was measured. Compound D inhibited the 3T3 cell growth expressing v1 (tumor) 101%.

(9) Kinase inhibitory effect by repeated administrations of polypeptide inhibitors of EML4-ALK fusion to 3T3 tumor expressing v1

The kinase inhibitory effect of compound C it was observed in a manner similar to that of Example 8 (8) with The following exceptions. 1 x 10 6 were inoculated 3T3 cell cells expressing v1 and administration of the Compound C was started 13 days after inoculation. The proof it was performed in the solvent group and the group of compound C, 3 Animals for each group. The administrations were carried out once a day for 3 days. The animals were dissected 4 hours after the last administration and the tumor was removed. Then, protein extracts were prepared from the tissues and immunoblotting was carried out using antibody phosphorylated anti-ALK. The results indicate that in the group of compound C, tyrosine autophosphorylation of EML4-ALK v1 fusion polypeptide in the tumor decreased significantly compared to the group of solvent From this result it was confirmed that the effect antitumor of compound C in the animal model described previously it was based on the kinase inhibitory effect of EML4-ALK v1 fusion polypeptide in the tumor.

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Example 9 EML4-ALK fusion polypeptide detection v1

A procedure was constructed to detect the EML4-ALK v1 fusion polypeptide in cells of the next way. 3T3 cells expressing v1 and cells were cultured U-87MG After washing 3 times with PBS, the cells were lysed with the lysis solution (Example 7 (1)). At 4 mg of the supernatant obtained after centrifugation, added anti-EML4 antibody (Cell Signaling Inc.) and It was reacted overnight at 4 ° C. So they were added G protein beads (Protein G Sepharose 4 Fast Flow; GE Healthcare Inc.) and immunoprecipitation was carried out for 2 hours. After centrifugation, the precipitates were washed 3 times with the wash solution (Example 7 (1)) and suspended in a solution that dissolved SDS. The supernatant was subjected to immunoblot using anti-ALK antibody. How result, the EML4-ALK v1 fusion polypeptide was detected in immunoprecipitates of 3T3 cells expressing v1, but it was not detected in U-87MG cells. Of the results described above it was possible to detect the presence of EML4-ALK v1 fusion polypeptide in cells cancerous and cancerous tissues that express the polypeptide of EML4-ALK v1 fusion using antibody anti-EML4 and anti-ALK antibody in combination, and it is clear that patients can be determined with cancer positive for the fusion polypeptide of EML4-ALK v1.

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Example 10 EML4-ALK fusion polypeptide analysis v2 (1) Construction of an expression vector of the EML4-ALK v2 fusion polypeptide

The vector in which the sequence of HindIII restriction enzyme cleavage at the end side 5 'of the initiation codon of the fusion polynucleotide of EML4-ALK v2 (EML4-ALKv2 / pCR2.1) se produced using the vector pCR2.1-TOPO in which it was cloned the EML4-ALK v2 fusion polynucleotide in the Example 4 (1).

The EML4-ALKv2 / pCR2.1 was digested with restriction enzyme HindIII, the fusion polynucleotide of EML4-ALK v2 was split, both ends were they turned blunt, then an adapter (adapter EcoRI-NotI-BamHI; Takara Bio Inc.) it was linked to both ends and the fragment was introduced at the site EcoRI of a pMXS retrovirus vector. This one was designated EML4-ALKv2 / pMXS.

Therefore, the EML4-ALKv2 / pCR2.1 was digested with restriction enzyme HindIII and XbaI, the EML4-ALK v2 fusion polynucleotide was cleaved and inserted into the HindIII / XbaI site of an expression vector pcDNA3.1 / Zeo (Invitrogen Inc.) which was modified so that the FLAG mark joined the N-terminus in the expression to produce a expression plasmid for the fusion polypeptide of EML4-ALK v2 to which the FLAG brand was attached to the end N (FLAG-EML4-ALKv2 / pcDNA3).

(2) Investigation of tumorigenicity of the polypeptide of EML4-ALK v2 fusion

He FLAG-EML4-ALKv2 / pcDNA3 (Example 10 (1)) was transfected into 3T3 cells using a reagent of transfection (FuGENE HD; Roche Diagnostics Inc.) according to the instruction attached. 3T3 cells that stably express the EML4-ALK v2 fusion polynucleotide se established by resistance to 80 µg / ml zeocin. The EML4-ALK fusion polynucleotide expression v2 in 3T3 cells was confirmed by immunoblot using anti-ALK antibody and antibody phosphorylated anti-ALK. 3T3 cells in which it expresses the EML4-ALK v2 fusion polypeptide se designate 3T3 cells that express v2. 3T3 cells that express v2 male BALB / c nude mice were inoculated subcutaneously 5 weeks old (Japan's Charles River Inc.) at 2 x 10 6 cells / mouse and were observed for 15 days. As in the case of the cells expressing v1 (the lower section of figure 3), it turned out that the tumor also formed in 3T3 cells that express the EML4-ALK v2 fusion polypeptide. The number of Tumor formation was 4 in 4.

From the previous results it was confirmed that the EML4-ALK v2 fusion polynucleotide, like that the EML4-ALK v1 fusion polynucleotide, it is also an oncogene that encodes polypeptides that have tumorigenicity for 3T3 cells.

(3) Screening of substances that inhibit the activity of intracellular autophosphorylation of the fusion polypeptide of EML4-ALK v2

To 293EBNA cells (Invitrogen Inc.) seeded in 24-well plates coated with collagen I (IWAKI; Asahi techno glass corp.) at 1 x 10 5 cells per well in DMEM medium containing 10% fetal bovine serum was introduced 100 FLAG-EMLA-ALKv2 / pcDNA3 ng (Example 10 (1) or pcDNA3 (white vector) as a control using a transfection reagent (lipofectamine 2000; Invitrogen Inc.). After cultivating for 20 hours, each of the compounds C or D or DMSO and the culture was incubated for 4 hours and Then the cells were recovered. Polypeptide expression of EML4-ALK v2 fusion and phosphorylation level of Tyrosine were measured by immunoblot using antibody anti-ALK and anti-ALK antibody phosphorylated

As a result, in the immunoblot using anti-ALK antibody was confirmed a band in the location of approximately 160 kDa in which it was expected that the fusion polynucleotide of EML4-ALK v2, and it was shown that the amount of expression of the fusion polynucleotide protein itself EML4-ALK v2 was almost constant in all samples. In addition, a band was confirmed at the same location in immunoblotting using anti-ALK antibody phosphorylated The amount of phosphorylation was calculated by quantifying in a similar way as in Example 7 (2). The speed of Phosphorylation inhibition by a compound was calculated from of the amount of phosphorylation when the compound was added, assuming the value when no compound was added (the compound solvent, DMSO) was 0% speed inhibition, and that the value when the white vector, pcDNA3, is introduced was 100% inhibition rate. The results indicate that each compound clearly inhibited kinase activity of the EML4-ALK v2 fusion polynucleotide in 293EBNA cells (Table 6). In a manner similar to that of Example 7 (4-2), except that compound A to D or DMSO were added to 3T3 cells expressing v2 (Example 10 (2-2)) at 10 µM or 10 nM, the amount of tyrosine phosphorylation of the fusion polypeptide of EML4-ALK v2 and the total amount of protein in the EML4-ALK v2 fusion polypeptide and the rate of inhibition of respective compounds in activity of intracellular kinase. Each compound clearly inhibited the kinase activity of the fusion polypeptide of EML4-ALK v2 in 3T3 cells expressing v2 (Table 6).

Compound A and B could be selected as substances that inhibited the activity of the fusion polypeptide of EML4-ALK v2 50% or more at a concentration of 10 µM or less, and compound C and D could be selected as substances that inhibited the activity of the fusion polypeptide of EML4-ALK v2 50% or more at a concentration of 0.1 µM or less. It is confirmed that the screening of the substances that inhibit the activity of the polypeptide of the present invention (cell based screening) can be performed using the polypeptide of EML4-ALK v2 fusion, in addition to the polypeptide of EML4-ALK v1 fusion.

TABLE 6

6

(4) Inhibitory effect of polypeptide inhibitors of EML4-ALK fusion on growth independent of anchoring 3T3 cells expressing v2

By a similar manner described as in Example 8 (3), the inhibitory effect of EML4-ALK fusion polypeptide inhibitors on cell growth independent of anchoring was investigated using 3T3 cells expressing v2 (Example 10 (2-2 )). The results showed that all compounds had growth inhibitory activity in 3T3 cells expressing v2. The rate of inhibition of the compounds was calculated assuming that the number of cells on day 0 and day 3 were 100% inhibition and 0% inhibition, respectively.
(Table 7).

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TABLE 7

7

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The results indicated that the compounds A-D inhibited independent cell growth of anchoring 3T3 cells expressing v2 inhibiting activity EML4-ALK fusion polypeptide kinase v2.

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Example 11 EML4-ALK fusion polypeptide analysis v3 (1) Detection of fusion polynucleotide from EML4-ALK v3 in clinical specimens and cloning of EML4-ALK v3 fusion polynucleotide

PCR (after 50 ° C for 2 minutes and 95 ° C for 15 minutes, 50 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds and 72 ° C for 90 seconds) was carried out using 75 cDNAs separated from clinical specimens and a kit of Quantitative PCR as in Example 3 (1) and oligonucleotides of SEQ ID NO: 131 and 82 as primers. As a result, in two cases a sequence of 515 bp (SEQ ID NO: 86) or 548 bp (SEQ ID was confirmed NO: 90) made by the merger of a part of EML4 and a part of ALK, which has a different melting point of the fusion polynucleotide of EML4-ALK v1 and fusion polynucleotide of EML4-ALK v2, was amplified using the cDNA of a patient shown to be positive for a PCR product described above as template and oligonucleotides of SEQ ID NO: 94 and 46 as primers, the PCR (35 cycles of 98 ° C for 10 seconds and 68 ° C for 6 minutes) was carried out with a DNA polymerase (PrimeStar HS DNA polymerase, Takara Bio Inc.). He PCR product was cloned into vector pT7Blue-2. As a result, the new polynucleotide was obtained (SEQ ID NO: 129) which was constituted by 700 bases from the initiation codon EMG4 gene ATG to intron portion 6 and 1691 bases from the exon 21 of the ALK gene to the termination codon of exon 30. The plasmid in which the cDNA of SEQ ID NO: 129 was cloned was designated EML4-ALKv3 / pT7Blue-2.

(2) Investigation of tumorigenicity of the polypeptide of EML4-ALK v3 fusion

Using the EML4-ALKv3 / pT7Blue-2, the vector of EML4-ALK v3 fusion polypeptide expression (designated EML4-ALKv3 / pMXS) was built according to a procedure of Example 4 (1). Therefore, using the EML4-ALKv3 / pT7Blue-2, a vector of expression (designated FLAG-EML4-ALKv3 / pMX-iresCD8) which expresses both the fusion polypeptide of EML4-ALK v3 that has the FLAG mark on the N-end as CD8, it was constructed using a similar procedure of Example 4 (1).

3T3 cells transfected with the EML4-ALKv3 / pMXS described above were inoculated subcutaneously to nude mice at 5 x 10 6 } cells / mouse and were observed after 20 days. It turned out that The tumor formed. Because the fusion polypeptide of EML4-ALK v3 was tumorigenic, it was indicated that the EML4-ALK v3 fusion polynucleotide is the gene Cause of cancer as well as v1 and v2. Hereinafter, the cells 3T3 in which the fusion polypeptide of EML4-ALK v3 designates 3T3 cells that express v3

(3) Procedure to detect polynucleotide mRNA EML4-ALK v3 fusion

Using 1 ng of the plasmid expressing the EML4-ALK v3 as a mold, and (SEQ ID NO: 132 and SEQ ID NO: 62, SEQ ID NO: 133 and SEQ ID NO: 64, SEQ ID NO: 134 and SEQ ID NO: 66, SEQ ID NO: 135 and SEQ ID NO: 68, SEQ ID NO: 136 and SEQ ID NO: 70, SEQ ID NO: 137 and SEQ ID NO: 72, SEQ ID NO: 138 and SEQ ID NO: 74, SEQ ID NO: 139 and SEQ ID NO: 76, SEQ ID NO: 140 and SEQ ID NO: 78, and SEQ ID NO: 141 and SEQ ID NO: 80) as a set of primers, the PCR (30 94 ° C cycles for 15 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute) was carried out using a DNA polymerase (DNA rTaq polymerase; Takara Bio Inc.). As a result, throughout the set of primers a DNA fragment was amplified single chain, each having the expected size. Of the previous results it was proved that the detection of the presence of EML4-ALK v3 fusion polynucleotide is possible carrying out RT-PCR according to the present example using mRNA extracted from samples of test subjects as mold.

(4) Screening procedure for inhibitors of EML4-ALK v3 fusion polypeptide

According to the procedure of Example 1, Example 5 (1) and Example 7 (1), the fusion polypeptide of EML4-ALK v3 was prepared using FLAG-EML4-ALKv3 / pMX-iresCD8.

The in vitro kinase activity of the purified EML4-ALK v3 fusion polypeptide as described above can be detected using a procedure of Example 7 (2). The inhibitory effect of AD compounds against in vitro kinase activity of the EML4-ALK v3 fusion polypeptide was investigated using the procedure according to the procedure of Example 7 (3). As a result, it was found that AD compounds inhibited autophosphorylation and phosphorylation activity in the peptide substrate by the purified EML4-ALK v3 fusion polypeptide (Table 8). These compounds could be selected as substances that inhibited the activity of the EML4-ALK v3 fusion polypeptide.

TABLE 8

8

The above results indicated that screening (an in vitro type screening) for a substance that inhibits the activity of the polypeptide of the present invention could be performed by preparing the EML4-ALK v3 fusion polypeptide and using the in vitro kinase activity as an index .

(5) Screening of substances that inhibit an activity of intracellular autophosphorylation of the fusion polypeptide of EML4-ALK v3

DMSO (control) or compound was added A-D to 3T3 cells expressing v3 (Example 11 (2)) at 10 µM or 10 nM and the amount of tyrosine phosphorylation of the fusion polypeptide of EML4-ALK v3 and the total amount of protein in the EML4-ALK v3 fusion polypeptide and velocity of inhibition of respective compounds in kinase activity intracellular was calculated in a manner similar to that of Example 7 (4-2). As a result, in the immunoblot using anti-ALK antibody, the bands were confirmed at the location of approximately 90 kDa in which the fusion polynucleotide of EML4-ALK v3 at the almost constant level in all samples. In addition, the bands were confirmed in the same location on immunoblot using antibody phosphorylated anti-ALK.

Each compound clearly inhibited the activity EML4-ALK v3 fusion polypeptide kinase in 3T3 cells expressing v3 (Table 9). It was confirmed that the screening of substances that inhibit polypeptide activity of the present invention (cell based screening) can be performed using the fusion polypeptide of EML4-ALK v3.

TABLE 9

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9

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(6) Inhibitory effect of polypeptide inhibitors of EML4-ALK fusion on growth independent of the anchor of 3T3 cells expressing v3

By a similar way described as in the Example 8 (3), the inhibitory effect of inhibitors (compound A-D) of the fusion polypeptide of EML4-ALK on the independent growth of Cell anchoring was investigated using 3T3 cells expressing v3 (Example 11 (2)). The results showed that all the compounds had growth inhibitory activity on 3T3 cells that Express v3 (Table 10).

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TABLE 10

10

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The previous results indicate that the compound A-D inhibited cell growth independent of the anchoring of 3T3 cells expressing v3 by inhibiting the kinase activity of the fusion polypeptide of EML4-ALK v3.

(7) Expression and cloning of polypeptide mRNA of EML4-ALK v3 fusion in cells NCI-H2228

Using the cDNA that was prepared by reverse transcription of the total NCI-H2228 cell RNA, a human non-small cell lung cancer cell line (American Type Culture Collection) as a template, the PCR (35 cycles of 98 ° C during 10 seconds, 68 ° C for 4 minutes) was carried out using oligonucleotides represented by SEQ ID NO: 94 and SEQ ID NO: 46 as primers and a DNA polymerase (primeSTAR HS DNA polymerase, Takara Bio Inc.). Using a part of the PCR product as a template, the PCR (35 cycles of 98 ° C for 10 seconds, 68 ° C for 4 minutes) was carried out using an oligonucleotide represented by SEQ ID NO: 105 that was provided with the cleavage sequence of HindIII restriction enzyme on the 5 'end side of the ATG initiation codon of the EML4 gene and an oligonucleotide represented by SEQE ID NO: 106 that was provided with the restriction enzyme cleavage sequence Xba I on the end side 3 'of the TGA termination codon of the ALK gene as primers and a DNA polymerase ( Pyrobest DNA polymerase; Takara Bio Inc.). The PCR product of approximately 2400 bp was obtained. This product was cloned by TA into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen Inc.) and the DNA sequence was analyzed. As a result, two types of polynucleotide were identified. One was a 2391 bp sequence (SEQ ID NO: 107) that was different in 4 bases, namely, bases 685, 903, 2000 and 2115 of SEQ ID NO: 129 (Example 11 (1)). The other was the 2358 bp sequence (SEQ ID NO: 109) that consisted of 667 bases from the ATG initiation codon of the EML4 gene to exon 6 and 1691 bases from exon 21 of the ALK gene to the termination codon of the exon 30. The portion corresponding to this ALK gene was approximately 1 base different from the sequence (4080-5770) of NM_004304 recorded in Genbank, without amino acid variation. Therefore, it was suggested that the differences in the portion of the ALK gene between the polynucleotide (SEQ ID NO: 129) identified in patients with lung cancer and the polynucleotide (SEQ ID NO: 107) cloned from NCI-H2228 were the
polymorphism.

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Neither the fusion polynucleotide of EML4-ALK v1 nor the fusion polynucleotide of EML4-ALK v2 was expressed endogenously in cells NCI-H2228.

(8) Screening of substances that inhibit an activity of intracellular autophosphorylation of the fusion polypeptide of EML4-ALK in NCI-H2228 cells

DMSO (control) or compound C was added to NCI-H2228 cells at 100 nM as the final concentration and the amount of tyrosine phosphorylation of the EML4-ALK fusion polypeptide and the rate of inhibition of compound C in autophosphorylation activity were calculated in a manner similar to that of Example 11 (5). Compound C inhibited the kinase activity of the EML4-ALK fusion polypeptide in 81% NCI-H2228 cells. It is confirmed that screening of substances that inhibit the activity of the EML4-ALK fusion polypeptide of the present invention (cell-type screening) can be performed using as an index autophosphorylation in NCI cells.
H2228

(9) Inhibitory effect of polypeptide inhibitors of EML4-ALK fusion on growth Anchor independent of NCI-H2228 cells

DMSO (control) or compound was added A-D to NCI-H2228 cells at 10 µM or 10 nM and this investigation was conducted in a similar way to that described in Example 8 (3), except that the cells are cultured for 5 days after drug addition. The results showed that compound A-D had growth inhibitory activity on cells NCI-H2228. The speed of inhibition of compounds was calculated assuming that the number of cells in the day 0 and on day 5 it was 100% inhibition and 0% inhibition, respectively (Table 11).

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TABLE 11

12

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The previous results indicate that the compound A-D inhibited cell growth anchor independent of NCI-H2228 cells non-small cell lung cancer, inhibiting the activity of EML4-ALK fusion polypeptide kinase. From the previous results it is clear that the inhibitor against EML4-ALK fusion polynucleotide is useful as therapeutic agent for positive lung cancer patients for the EML4-ALK fusion polynucleotide.

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References cited in the description

This list of references cited by the applicant is intended solely to help the reader and not It is part of the European patent document. Although it has been put maximum care in its realization, errors cannot be excluded or omissions and the EPO declines any responsibility to respect.

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         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tgagcttgac aaagtggtcg

\hfill

20

 \ hskip-.1em \ dddseqskip 

tgagcttgac aaagtggtcg

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gggaaggtga aggtcgga

\hfill

18

 \ hskip-.1em \ dddseqskip 

gggaaggtga aggtcgga

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gcagccctgg tgaccag

\hfill

17

 \ hskip-.1em \ dddseqskip 

gcagccctgg tgaccag

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 247

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

         \ hskip1cm
      

53

         \ vskip0.400000 \ baselineskip
      

<210> 15

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 15

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gcacagggaa ataagc

\hfill

16

 \ hskip-.1em \ dddseqskip 

gcacagggaa ataagc

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 16

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gggcagagtc atgtta

\hfill

16

 \ hskip-.1em \ dddseqskip 

gggcagagtc atgtta

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 17

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccagtagtac tctcag

\hfill

16

 \ hskip-.1em \ dddseqskip 

ccagtagtac tctcag

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 18

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cccctgagct ctgaac

\hfill

16

 \ hskip-.1em \ dddseqskip 

cccctgagct ctgaac

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccagtgaaca cagttgt

\hfill

17

 \ hskip-.1em \ dddseqskip 

ccagtgaaca cagttgt

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 20

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 20

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cctggatete catatcc

\hfill

17

 \ hskip-.1em \ dddseqskip 

cctggatete catatcc

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 21

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gatgtaagtg gagacag

\hfill

17

 \ hskip-.1em \ dddseqskip 

gatgtaagtg gagacag

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 22

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 22

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ttcctacagg aagcctc

\hfill

17

 \ hskip-.1em \ dddseqskip 

ttcctacagg aagcctc

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 23

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 23

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gataettate tacctatg

\hfill

18

 \ hskip-.1em \ dddseqskip 

gataettate tacctatg

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 24

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cctttccatc atacttag

\hfill

18

 \ hskip-.1em \ dddseqskip 

cctttccatc atacttag

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 25

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 25

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gatgataaat attgatgt

\hfill

18

 \ hskip-.1em \ dddseqskip 

gatgataaat attgatgt

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 26

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 26

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aagcactaca caggccac

\hfill

18

 \ hskip-.1em \ dddseqskip 

aagcactaca caggccac

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 27

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> RNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tgtcctccct ctcgtggta

\hfill

19

 \ hskip-.1em \ dddseqskip 

tgtcctccct ctcgtggta

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 28

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 28

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gattaaagaa ggtgtgtct

\hfill

19

 \ hskip-.1em \ dddseqskip 

gattaaagaa ggtgtgtct

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 29

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 29

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggctactctt gttagtttg

\hfill

19

 \ hskip-.1em \ dddseqskip 

ggctactctt gttagtttg

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 30

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 30

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtctggttcc tccaagaag

\hfill

19

 \ hskip-.1em \ dddseqskip 

gtctggttcc tccaagaag

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 31

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 31

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttacatgat accttcaggc

\hfill

20

 \ hskip-.1em \ dddseqskip 

cttacatgat accttcaggc

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 32

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 32

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

caagaagcag actggagatg

\hfill

20

 \ hskip-.1em \ dddseqskip 

caagaagcag actggagatg

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 33

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 33

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ttgacctgaa cagcaagttt

\hfill

20

 \ hskip-.1em \ dddseqskip 

ttgacctgaa cagcaagttt

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 34

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 34

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggtgggtggt cagctgcaac

\hfill

20

 \ hskip-.1em \ dddseqskip 

ggtgggtggt cagctgcaac

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 35

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 35

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

agctacatca cacaccttga ctgg

\hfill

24

 \ hskip-.1em \ dddseqskip 

agctacatca cacaccttga ctgg

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 36

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 36

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

agcttgctca gcttgtactc aggg

\hfill

24

 \ hskip-.1em \ dddseqskip 

agcttgctca gcttgtactc aggg

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 37

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 37

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttccatggg aatcag

\hfill

16

 \ hskip-.1em \ dddseqskip 

cttccatggg aatcag

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 38

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 38

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ttgcagctcc tggtgc

\hfill

16

 \ hskip-.1em \ dddseqskip 

ttgcagctcc tggtgc

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 39

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 39

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtagaacaga aggcac

\hfill

16

 \ hskip-.1em \ dddseqskip 

gtagaacaga aggcac

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 40

         \ vskip0.400000 \ baselineskip
      

<211> 224

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 40

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccacacctgg gaaaggacct aaag

\hfill

24

 \ hskip-.1em \ dddseqskip 

ccacacctgg gaaaggacct aaag

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 41

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 41

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

atgctgctct gaatggt

\hfill

17

 \ hskip-.1em \ dddseqskip 

atgctgctct gaatggt

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 42

         \ vskip0.400000 \ baselineskip
      

<211> 23

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 42

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aagcagtggt atcaacgcag agt

\hfill

23

 \ hskip-.1em \ dddseqskip 

aagcagtggt atcaacgcag agt

 \ hfill 

2. 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 43

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 43

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggcagggagg aatatga

\hfill

17

 \ hskip-.1em \ dddseqskip 

ggcagggagg aatatga

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 44

         \ vskip0.400000 \ baselineskip
      

<211> 22

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 44

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

caattacggc taccagcaac ag

\hfill

22

 \ hskip-.1em \ dddseqskip 

caattacggc taccagcaac ag

 \ hfill 

22

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 45

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 45

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gatagatggg catttatg

\hfill

18

 \ hskip-.1em \ dddseqskip 

gatagatggg catttatg

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 46

         \ vskip0.400000 \ baselineskip
      

<211> 23

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 46

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccacggtctt agggatccca agg

\hfill

23

 \ hskip-.1em \ dddseqskip 

ccacggtctt agggatccca agg

 \ hfill 

2. 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 47

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 47

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gggaatcagtttgtagtt

\hfill

18

 \ hskip-.1em \ dddseqskip 

gggaatcagtttgtagtt

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 48

         \ vskip0.400000 \ baselineskip
      

<211> 23

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 48

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tatcctcgta atgaccagct cca

\hfill

23

 \ hskip-.1em \ dddseqskip 

tatcctcgta atgaccagct cca

 \ hfill 

2. 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 49

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 49

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tacttctcct ggaggcagg

\hfill

19

 \ hskip-.1em \ dddseqskip 

tacttctcct ggaggcagg

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 50

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Mus musculus

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 50

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

caggagagaa aggatttggc taca

\hfill

24

 \ hskip-.1em \ dddseqskip 

caggagagaa aggatttggc taca

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 51

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 51

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tacaagtaaa cgtggctag

\hfill

19

 \ hskip-.1em \ dddseqskip 

tacaagtaaa cgtggctag

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 52

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 52

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttccggcgg tacactgca

\hfill

19

 \ hskip-.1em \ dddseqskip 

cttccggcgg tacactgca

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 53

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 53

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ctgttaaagt gaacactttc

\hfill

20

 \ hskip-.1em \ dddseqskip 

ctgttaaagt gaacactttc

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 54

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 54

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

acatggcctg gcagcctggc

\hfill

20

 \ hskip-.1em \ dddseqskip 

acatggcctg gcagcctggc

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 55

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 55

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtgtcttaat gtttttcagt

\hfill

20

 \ hskip-.1em \ dddseqskip 

gtgtcttaat gtttttcagt

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 56

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Mus musculus

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 56

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tccaccctgg atcatgaagt c

\hfill

21

 \ hskip-.1em \ dddseqskip 

tccaccctgg atcatgaagt c

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 57

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 57

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggggaattca tggacggttt cgccggc

\hfill

27

 \ hskip-.1em \ dddseqskip 

ggggaattca tggacggttt cgccggc

 \ hfill 

27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 58

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 58

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cggcggtaca agtacaatat ttcatagtct

\hfill

30

 \ hskip-.1em \ dddseqskip 

cggcggtaca agtacaatat ttcatagtct

 \ hfill 

30

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 59

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 59

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aaggaattcg gtttcgccgg cagtctc

\hfill

27

 \ hskip-.1em \ dddseqskip 

aaggaattcg gtttcgccgg cagtctc

 \ hfill 

27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 60

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 60

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

agagaattca gtgtgcgacc gagctca

\hfill

27

 \ hskip-.1em \ dddseqskip 

agagaattca gtgtgcgacc gagctca

 \ hfill 

27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 61

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 61

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tggagcggca attcac

\hfill

16

 \ hskip-.1em \ dddseqskip 

tggagcggca attcac

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 62

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 62

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aaagcagtag ttgggg

\hfill

16

 \ hskip-.1em \ dddseqskip 

aaagcagtag ttgggg

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 63

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 63

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggggaatgga gatgtt

\hfill

16

 \ hskip-.1em \ dddseqskip 

ggggaatgga gatgtt

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 64

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 64

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tttccgcggc acctcc

\hfill

16

 \ hskip-.1em \ dddseqskip 

tttccgcggc acctcc

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 65

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 65

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ctaacaagaa aacaggg

\hfill

17

 \ hskip-.1em \ dddseqskip 

ctaacaagaa aacaggg

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 66

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 66

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttctggacc tggagcg

\hfill

17

 \ hskip-.1em \ dddseqskip 

cttctggacc tggagcg

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 67

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 67

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttctggacc tggagcg

\hfill

17

 \ hskip-.1em \ dddseqskip 

cttctggacc tggagcg

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 68

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 68

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttctggacc tggagcg

\hfill

17

 \ hskip-.1em \ dddseqskip 

cttctggacc tggagcg

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 69

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 69

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggctgtggag tttcaccc

\hfill

18

 \ hskip-.1em \ dddseqskip 

ggctgtggag tttcaccc

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 70

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 70

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

agctcctggt gcttccgg

\hfill

18

 \ hskip-.1em \ dddseqskip 

agctcctggt gcttccgg

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 71

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 71

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cccaacagat gcaaatac

\hfill

18

 \ hskip-.1em \ dddseqskip 

cccaacagat gcaaatac

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 72

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 72

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tcagggctct gcagctcc

\hfill

18

 \ hskip-.1em \ dddseqskip 

tcagggctct gcagctcc

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 73

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 73

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gacaacaaat gaagttgtt

\hfill

19

 \ hskip-.1em \ dddseqskip 

gacaacaaat gaagttgtt

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 74

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 74

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

atctgcatgg cttgcagct

\hfill

19

 \ hskip-.1em \ dddseqskip 

atctgcatgg cttgcagct

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 75

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 75

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccataattac atgcggtaa

\hfill

19

 \ hskip-.1em \ dddseqskip 

ccataattac atgcggtaa

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 76

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 76

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gagcttgctc agcttgtac

\hfill

19

 \ hskip-.1em \ dddseqskip 

gagcttgctc agcttgtac

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 77

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 77

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtgtttagca ttcttgggga

\hfill

20

 \ hskip-.1em \ dddseqskip 

gtgtttagca ttcttgggga

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 78

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 78

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gaccccgaat gagggtgatg

\hfill

20

 \ hskip-.1em \ dddseqskip 

gaccccgaat gagggtgatg

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 79

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 79

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gggaattttt gggaaatatg

\hfill

20

 \ hskip-.1em \ dddseqskip 

gggaattttt gggaaatatg

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 80

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 80

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cctccttcag gtcactgatg

\hfill

20

 \ hskip-.1em \ dddseqskip 

cctccttcag gtcactgatg

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 81

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 81

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cctggctgta ggatct

\hfill

16

 \ hskip-.1em \ dddseqskip 

cctggctgta ggatct

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 82

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 82

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tcttgccagc aaagcagtag ttgg

\hfill

24

 \ hskip-.1em \ dddseqskip 

tcttgccagc aaagcagtag ttgg

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 83

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 83

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtggccatag gaacgc

\hfill

16

 \ hskip-.1em \ dddseqskip 

gtggccatag gaacgc

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 84

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 84

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtgcttccgg cggtac

\hfill

16

 \ hskip-.1em \ dddseqskip 

gtgcttccgg cggtac

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 85

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 85

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ctcaatagat ggtacct

\hfill

17

 \ hskip-.1em \ dddseqskip 

ctcaatagat ggtacct

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 86

         \ vskip0.400000 \ baselineskip
      

<211> 515

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 86

         \ hskip1cm
      

54

         \ vskip0.400000 \ baselineskip
      

<210> 87

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 87

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gctctctgtg atgcgct

\hfill

17

 \ hskip-.1em \ dddseqskip 

gctctctgtg atgcgct

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 88

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 88

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggtgcggagc ttgctca

\hfill

17

 \ hskip-.1em \ dddseqskip 

ggtgcggagc ttgctca

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 89

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 89

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

caggcaggtg gtttgttc

\hfill

18

 \ hskip-.1em \ dddseqskip 

caggcaggtg gtttgttc

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 90

         \ vskip0.400000 \ baselineskip
      

<211> 548

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 90

         \ hskip1cm
      

55

         \ vskip0.400000 \ baselineskip
      

<210> 91

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 91

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggaatgaaca gctctctg

\hfill

18

 \ hskip-.1em \ dddseqskip 

ggaatgaaca gctctctg

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 92

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 92

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tcggtcatga tggtcgag

\hfill

18

 \ hskip-.1em \ dddseqskip 

tcggtcatga tggtcgag

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 93

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 93

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggatgcagaa accagagat

\hfill

19

 \ hskip-.1em \ dddseqskip 

ggatgcagaa accagagat

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 94

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 94

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

actctgtcgg tccgctgaat gaag

\hfill

24

 \ hskip-.1em \ dddseqskip 

actctgtcgg tccgctgaat gaag

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 95

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> RNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 95

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggaacgcact caggcaggt

\hfill

19

 \ hskip-.1em \ dddseqskip 

ggaacgcact caggcaggt

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 96

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 96

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ctcagggctc tgcagctcc

\hfill

19

 \ hskip-.1em \ dddseqskip 

ctcagggctc tgcagctcc

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 97

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 97

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ctatccacac agacgggaat

\hfill

20

 \ hskip-.1em \ dddseqskip 

ctatccacac agacgggaat

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 98

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 98

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gcttgtactc agggctctgc

\hfill

20

 \ hskip-.1em \ dddseqskip 

gcttgtactc agggctctgc

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 99

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 99

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gtagtctctg aaaatggaag

\hfill

20

 \ hskip-.1em \ dddseqskip 

gtagtctctg aaaatggaag

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 100

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 100

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tcttgccagc aaagcagtag

\hfill

20

 \ hskip-.1em \ dddseqskip 

tcttgccagc aaagcagtag

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 101

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 101

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

atattgtact tgtaccgccg gaagcaccag

\hfill

30

 \ hskip-.1em \ dddseqskip 

atattgtact tgtaccgccg gaagcaccag

 \ hfill 

30

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 102

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 102

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gggtctagat cagggcccag gctggtt

\hfill

27

 \ hskip-.1em \ dddseqskip 

gggtctagat cagggcccag gctggtt

 \ hfill 

27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 103

         \ vskip0.400000 \ baselineskip
      

<211> 35

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 103

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccctgcaagt ggctgtgatg acgctgcctg aagtg

\hfill

35

 \ hskip-.1em \ dddseqskip 

ccctgcaagt ggctgtgatg acgctgcctg aagtg

 \ hfill 

35

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 104

         \ vskip0.400000 \ baselineskip
      

<211> 35

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 104

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cacttcaggc agcgtcatca cagccacttg caggg

\hfill

35

 \ hskip-.1em \ dddseqskip 

cacttcaggc agcgtcatca cagccacttg caggg

 \ hfill 

35

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 105

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 105

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aagcttatgg acggttícgc cggcagtctc

\hfill

30

 \ hskip-.1em \ dddseqskip 

aagcttatgg acggttícgc cggcagtctc

 \ hfill 

30

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 106

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized primer sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 106

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tctagatcag ggcccaggct ggtt

\hfill

24

 \ hskip-.1em \ dddseqskip 

tctagatcag ggcccaggct ggtt

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 107

         \ vskip0.400000 \ baselineskip
      

<211> 2391

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (2388)

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 107

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

56

         \ hskip1cm
      

57

         \ hskip1cm
      

58

         \ hskip1cm
      

59

         \ hskip1cm
      

60

         \ vskip0.400000 \ baselineskip
      

<210> 108

         \ vskip0.400000 \ baselineskip
      

<211> 796

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 108

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

62

         \ hskip1cm
      

63

         \ hskip1cm
      

64

         \ hskip1cm
      

65

         \ hskip1cm
      

66

         \ vskip0.400000 \ baselineskip
      

<210> 109

         \ vskip0.400000 \ baselineskip
      

<211> 2358

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (2355)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 109

         \ hskip1cm
      

67

         \ hskip1cm
      

68

         \ hskip1cm
      

69

         \ hskip1cm
      

70

         \ hskip1cm
      

71

         \ vskip0.400000 \ baselineskip
      

<210> 110

         \ vskip0.400000 \ baselineskip
      

<211> 785

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 110

         \ hskip1cm
      

72

         \ hskip1cm
      

73

         \ hskip1cm
      

74

         \ hskip1cm
      

75

         \ hskip1cm
      

76

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 111

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-1

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 111

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ugggaaagga ccuaaagtgt a

\hfill

21

 \ hskip-.1em \ dddseqskip 

ugggaaagga ccuaaagtgt a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 112

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-1

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 112

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cactttaggu ccuuucccag g

\hfill

21

 \ hskip-.1em \ dddseqskip 

cactttaggu ccuuucccag g

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 113

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-2

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 113

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gggaaaggac cuaaagtgta c

\hfill

21

 \ hskip-.1em \ dddseqskip 

gggaaaggac cuaaagtgta c

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 114

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-2

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 114

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

acacttuagg uccuuuccca g

\hfill

21

 \ hskip-.1em \ dddseqskip 

acacttuagg uccuuuccca g

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 115

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-3

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 115

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggaccuaaag uguaccgccg g

\hfill

21

 \ hskip-.1em \ dddseqskip 

ggaccuaaag uguaccgccg g

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 116

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-3

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 116

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggcggtacac uuuagguccu u

\hfill

21

 \ hskip-.1em \ dddseqskip 

ggcggtacac uuuagguccu u

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 117

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-4

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 117

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ccuaaagugu accgccggaa g

\hfill

21

 \ hskip-.1em \ dddseqskip 

ccuaaagugu accgccggaa g

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 118

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-4

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA ...

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 118

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tccggcggua cacuuuaggu c

\hfill

21

 \ hskip-.1em \ dddseqskip 

tccggcggua cacuuuaggu c

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 119

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-5

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 119

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aaaguguacc gccggaagca c

\hfill

21

 \ hskip-.1em \ dddseqskip 

aaaguguacc gccggaagca c

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 120

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-5

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 120

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gcttccggcg guacacuuua g

\hfill

21

 \ hskip-.1em \ dddseqskip 

gcttccggcg guacacuuua g

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 121

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-6

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 121

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aaguguaccg ccggaagcac c

\hfill

21

 \ hskip-.1em \ dddseqskip 

aaguguaccg ccggaagcac c

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 122

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-6

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mist_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 122

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tgcttccggc gguacacuuu a

\hfill

21

 \ hskip-.1em \ dddseqskip 

tgcttccggc gguacacuuu a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 123

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-7

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 123

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggccuguaua ccggataatg a

\hfill

21

 \ hskip-.1em \ dddseqskip 

ggccuguaua ccggataatg a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 124

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-7

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 124

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

attatccggu auacaggccc a

\hfill

21

 \ hskip-.1em \ dddseqskip 

attatccggu auacaggccc a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 125

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-8

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 125

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ggccuguaua ccggauaaug a

\hfill

21

 \ hskip-.1em \ dddseqskip 

ggccuguaua ccggauaaug a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 126

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-8

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 126

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

auuauccggu auacaggccc a

\hfill

21

 \ hskip-.1em \ dddseqskip 

auuauccggu auacaggccc a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 127

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized transcription chain of SiRNA-9

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (13)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (14) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 127

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cggcugcaau cgattgatag c

\hfill

21

 \ hskip-.1em \ dddseqskip 

cggcugcaau cgattgatag c

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 128

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA / RNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Description of artificial sequence: an artificially synthesized complementary chain of SiRNA-9

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (6)

         \ vskip0.400000 \ baselineskip
      

<223> DNA

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> RNA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 128

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tatcaaucga uugcagccga a

\hfill

21

 \ hskip-.1em \ dddseqskip 

tatcaaucga uugcagccga a

 \ hfill 

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 129

         \ vskip0.400000 \ baselineskip
      

<211> 2391

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (2388)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 129

         \ hskip1cm
      

77

         \ hskip1cm
      

78

         \ hskip1cm
      

79

         \ hskip1cm
      

80

         \ hskip1cm
      

81

         \ hskip1cm
      

82

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 130

         \ vskip0.400000 \ baselineskip
      

<211> 796

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 130

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

83

         \ hskip1cm
      

84

         \ hskip1cm
      

85

         \ hskip1cm
      

86

         \ hskip1cm
      

87

         \ hskip1cm
      

88

         \ vskip0.400000 \ baselineskip
      

<210> 131

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 131 24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

taccagtgct gtctcaattg cagg

\hfill

24

 \ hskip-.1em \ dddseqskip 

taccagtgct gtctcaattg cagg

 \ hfill 

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 132

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 132 16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cacagacaaa ctccag

\hfill

16

 \ hskip-.1em \ dddseqskip 

cacagacaaa ctccag

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 133

         \ vskip0.400000 \ baselineskip
      

<211> 16

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 133

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cgaccatcac cagctg

\hfill

16

 \ hskip-.1em \ dddseqskip 

cgaccatcac cagctg

 \ hfill 

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 134

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 134

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

agaatgctac tcccacc

\hfill

17

 \ hskip-.1em \ dddseqskip 

agaatgctac tcccacc

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 135

         \ vskip0.400000 \ baselineskip
      

<211> 17

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 135

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cttcacaacc tctccaa

\hfill

17

 \ hskip-.1em \ dddseqskip 

cttcacaacc tctccaa

 \ hfill 

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 136

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 136

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aaccacaagg acagagag

\hfill

18

 \ hskip-.1em \ dddseqskip 

aaccacaagg acagagag

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 137

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 137

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aagtccacaa attcgagc

\hfill

18

 \ hskip-.1em \ dddseqskip 

aagtccacaa attcgagc

 \ hfill 

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 138

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 138

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

gaatctcatt ctaatgatc

\hfill

19

 \ hskip-.1em \ dddseqskip 

gaatctcatt ctaatgatc

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 139

         \ vskip0.400000 \ baselineskip
      

<211> 19

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 139

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

ttcgagcatc accttctcc

\hfill

19

 \ hskip-.1em \ dddseqskip 

ttcgagcatc accttctcc

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 140

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 140

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

aaagtcacat aattcttggg

\hfill

20

 \ hskip-.1em \ dddseqskip 

aaagtcacat aattcttggg

 \ hfill 

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 141

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 141

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

cccaccaaaa gcataaaacg

\hfill

20

 \ hskip-.1em \ dddseqskip 

cccaccaaaa gcataaaacg

 \ hfill 

twenty

Claims (17)

1. Procedure to detect a fusion gene of the protein-like gene 4 associated with microtubules echinoderms (EML4) and anaplastic lymphoma kinase (ALK) gene that comprises the step of detecting the presence of a polynucleotide in a sample obtained from a test subject, in which the polynucleotide encodes a polypeptide selected from the group that it is formed by:

(one)

a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and that has an activity of kinase,

(2)

a polypeptide comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 or 7 with the deletion, substitution and / or insertion of 1 to 10 amino acids and that It has a kinase activity,

(3)

a polypeptide comprising an amino acid sequence with 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 7 and having a kinase activity, and

(4)

a polypeptide that is constituted by the amino acid sequence represented by SEQ ID NO: 2, 7 or 130.

         \ vskip1.000000 \ baselineskip
      

2. Procedure to detect a protein from fusion encoded by a fusion gene of the EML4 gene and the ALK gene comprising the step of detecting the presence of a polypeptide in a sample obtained from a test subject, in which the Polypeptide is selected from the group consisting of:

(one)

a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and that has an activity of kinase,

(2)

a polypeptide comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 or 7 with the deletion, substitution and / or insertion of 1 to 10 amino acids and that It has a kinase activity,

(3)

a polypeptide comprising an amino acid sequence with 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 7 and having a kinase activity, and

(4)

a polypeptide that is constituted by the amino acid sequence represented by SEX ID NO: 2, 7 or 130.

         \ vskip1.000000 \ baselineskip
      

3. Procedure of claim 1 or the claim 2, wherein the polypeptide comprises the sequence of amino acids represented by SEQ ID NO: 2, 7 or 130 and which has a kinase activity. 4. Procedure of claim 1 or the claim 2, wherein the polypeptide is constituted by the amino acid sequence represented by SEQ ID NO: 2, 7 or 130. 5. Procedure of claim 1 or the claim 2, wherein the polypeptide comprises the sequence of amino acids represented by SEQ ID NO: 2 or 7 and which has a kinase activity. 6. Procedure of claim 1 or the claim 2, wherein the polypeptide is constituted by the amino acid sequence represented by SEQ ID NO: 2 or 7. 7. Kit for the detection of a fusion gene of EML4 gene and the ALK gene comprising sense and antisense primers that specifically amplify a polynucleotide encoding a polypeptide selected from the group consisting of:

(one)

a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and that has an activity of kinase,

(2)

a polypeptide comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 or 7 with the deletion, substitution and / or insertion of 1 to 10 amino acids and that It has a kinase activity,

(3)

a polypeptide comprising an amino acid sequence with 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 7 and having a kinase activity, and

(4)

a polypeptide that is constituted by the amino acid sequence represented by SEQ ID NO: 2, 7 or 130.

         \ vskip1.000000 \ baselineskip
      

8. Kit of claim 7, wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 2, 7 or 130 and that has a kinase activity. 9. Kit of claim 7, wherein the polypeptide is constituted by the amino acid sequence represented by SEQ ID NO: 2, 7 or 130. 10. Kit of claim 7, wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 2 or 7 and having a kinase activity. 11. Kit of claim 7, wherein the polypeptide is constituted by the amino acid sequence represented by SEQ ID NO: 2 or 7. 12. Set of primers to detect a gene of fusion of the EML4 gene and the ALK gene, in which the set of primers are selected from the following a) - c):

to)

a set of primers comprising a primer antisense that is made up of a nucleic acid molecule that hybridizes under restrictive conditions to a party that encodes ALK of a polynucleotide and a sense primer that is consisting of a nucleic acid molecule that hybridizes under restrictive conditions to the complementary chain of a party encoding EML4 of the polynucleotide, in which the polynucleotide encodes a polypeptide selected from the group that is constituted by:

(one)

a polypeptide comprising the sequence of amino acids represented by SEQ ID NO: 2 or 7 and which has a kinase activity,

(2)

a polypeptide comprising a sequence of amino acids derived from the amino acid sequence represented by SEQ ID NO: 2 or 7 with the deletion, replacement and / or insertion of 1 to 10 amino acids and which has a kinase activity,

(3)

a polypeptide comprising a sequence of amino acids with 90% or more identity with the sequence of amino acids represented by SEQ ID NO: 2 or 7 and which has a kinase activity, and

(4)

a polypeptide that is constituted by the sequence of amino acids represented by SEQ ID NO: 2 or 7;

b)

a set of primers comprising a primer antisense that is made up of a nucleic acid molecule that hybridizes under restrictive conditions to a party that encodes ALK of the polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 4 and a primer sense that is constituted by a nucleic acid molecule that hybridizes under restrictive conditions to the complementary chain of a part encoding EML4 of the polynucleotide; Y

C)

a set of primers comprising a primer antisense that is made up of a nucleic acid molecule that hybridizes under restrictive conditions to a party that encodes ALK of the polynucleotide that is constituted by the nucleotide sequence represented by SEQ ID NO: 5 and a primer sense that is constituted by a nucleic acid molecule that hybridizes under restrictive conditions to the complementary chain of a part encoding EML4 of the polynucleotide.

         \ vskip1.000000 \ baselineskip
      

13. Set of primers of the claim 12, wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 2 or 7 and that has an activity of kinase 14. Set of primers of the claim 12, in which the polypeptide is constituted by the sequence of amino acids represented by SEQ ID NO: 2 or 7. 15. Set of primers of a felt primer comprising an oligonucleotide with at least all 16 bases consecutive ones located in the number of bases 1 to 2242 in SEQ ID NO: 6 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least all 16 bases consecutive ones located in the number of bases 2243 to 3933 in SEQ ID NO: 6, or a set of primers consisting of strings complementary to them, in which the primers felt and antisense give amplification products of 1 kb or less than size. 16. Set of primers of a felt primer comprising an oligonucleotide with at least all 16 bases consecutive ones located in the number of bases 1 to 3629 in SEQ ID NO: 4 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least all 16 bases consecutive ones located in the number of bases 3630 to 3979 in SEQ ID NO: 4, or a set of primers consisting of strings complementary to them, in which the primers felt and antisense give amplification products of 1 kb or less than size. 17. Set of primers of a felt primer comprising an oligonucleotide with at least all 16 bases consecutive ones located in the number of bases 1 to 579 in SEQ ID NO: 5 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least all 16 bases consecutive ones located in the number of bases 580 to 853 in SEQ ID NO: 5, or a set of primers consisting of strings complementary to them.